Organic anion transporter genes and proteins

ABSTRACT

This invention relates to novel human organic anion transporter polypeptides and genes encoding such polypeptides. The invention is directed towards the isolation, characterization and use of human organic anion transporter polypeptides for pharmacological screening of substrates, analogues of substrates, and modulators of human organic anion proteins. This invention also provides cell cultures, which stably contain nucleic acid sequences encoding human organic anion transport polypeptides. Such cell lines are useful for screening compounds in vitro to identify and characterize modulators, inhibitors and substrates of human organic anion transporter polypeptides.

RELATED APPLICATIONS

This application claims the benefit of U.S. Ser. No. 60/143,771,entitled “Organic Anion Transporter Genes and Proteins,” which was filedJul. 12, 1999 and which is incorporated by reference herein in itsentirety, including figures.

FIELD OF THE INVENTION

The present invention relates to specific proteins as well asrecombinant versions of these proteins which are organic aniontransporters. These proteins include human membrane proteinspreferentially found in kidney and liver cells. The present inventionalso relates to nucleotide sequences encoding these novel organic aniontransporters. In another aspect, the present invention relates tomethods for using these proteins as in vitro screening agents toidentify substrates for the proteins and inhibitors which blocktransport activity. In yet another aspect, these proteins may be used inin vitro assays to predict drug pharmacokinetics, drug distribution anddrug toxicity. The invention also discloses antibodies that specificallybind to these proteins and which may be used in diagnostic assays.

BACKGROUND OF THE INVENTION

The following describes certain relevant art, none of which is admittedto be prior art to the inventions described herein.

The liver and kidney are two organs that can extract a variety oforganic anions from circulation, including endogenous compounds such asbile acids and bilirubin and xenobiotics such as sulfobromophthalein(BSP) and p-aminohippurate (PAH). The liver and kidney thus have thecritical functions of bile secretion, detoxification, and drugelimination. Since the cell membrane represents a hydrophobic barrierthat prevents influx of charged or hydrophilic molecules, hepatocytesand kidney epithelial cells express proteins on their basolateralmembranes to facilitate transport of organic anions into the cell. Theliver and kidney contain membrane proteins that transport very specificcompounds such as taurocholic acid or prostaglandins. However, they alsohave proteins that exhibit a wider substrate specificity. Recently,transporters of the latter type have been cloned from rat liver andkidney. One such protein is called the organic anion transportingpolypeptide, oatp (Jacquemin et al. (1994) PNAS 91:133-37). This proteinbelongs to a family of related transporters, members of which consistsof oatp1, oatp2, oatp3, and prostaglandin transporters. These proteinsshare homology at the amino acid level (Noe et al. (1997) PNAS 94:10346-50; Kanai et al. (1995) Science 268: 866-69; Abe et al. (1998) J.Biol. Chem. 273: 22395-401). When expressed in Xenopus oocytes or inmammalian cells, members of the oatp family were shown to transport manytypes of organic anions including taurocholic acid, BSP, and conjugatedsteroid hormones. The related prostaglandin transporters show highaffinity transport of prostaglandins.

Shortly after the cloning of oatp1, members of another organic aniontransporter family, OAT1 and OAT2, were isolated from rat kidney andliver, respectively (Sekine et al. (1997) J. Biol. Chem 272: 18526-29;Sekine et al. (1998) FEBS Letters 429: 179-82). Rat OAT1 and OAT2 arehomologous to each other but show no significant homology to members ofthe oatp family. However, they show some homology to the family oforganic cation transporters (OCTs), suggesting that these two familiesof proteins share a common origin. Rat OAT1 and OAT2 can transport manydifferent organic anions. OAT1, when expressed in oocytes, can transportp-aminohippurate (PAH), methotrexate, and glutarate. OAT1 is most likelythe molecular entity responsible for the classicalp-aminohippurate/α-ketoglutarate exchanger found in the kidney proximaltubule. OAT2, expressed predominantly in the liver, has been shown totransport organic anions such as salicylate, methotrexate, andα-ketoglutarate. Therefore, OAT1 and OAT2 belong to yet another familyof multi-specific organic anion transporters distinct from the oatpfamily.

SUMMARY OF THE INVENTION

The present invention is directed to a group of polypeptides,preferentially expressed in the liver and kidneys of humans and whichhave activity as organic anion transporters. We refer to thesepolypeptides as “human organic anion transporters” or “hOATpolypeptides”. These proteins and their properties are described indetail below.

The invention also concerns, nucleic acids encoding hOAT polypeptides,cells, tissues and animals containing such nucleic acids, antibodies tothe polypeptides, assays utilizing the polypeptides, and methodsrelating to all of the foregoing.

A first aspect of the invention features an isolated, enriched, orpurified nucleic acid molecule encoding an hOAT polypeptide or encodinga fragment of an hOAT polypeptide.

In preferred embodiments the isolated nucleic acid comprises a nucleicacid sequence set forth in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ IDNO:4, SEQ ID NO:5, SEQ ID NO:6, or a functional derivative thereof, anucleic acid sequence that hybridizes to the nucleic acid sequence setforth in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ IDNO:5, SEQ ID NO:6, or a functional derivative thereof.

The nucleic acid may be isolated from a natural source by cDNA cloningor subtractive hybridization; the natural source may be mammalian(human) blood, semen, or tissue and the nucleic acid may be synthesizedby the triester or other method or by using an automated DNAsynthesizer. Preferably, the nucleic acid is isolated from mammaliankidney or brain, most preferably from liver.

Individual clones isolated from a cDNA library may be purified toelectrophoretic homogeneity. The claimed DNA molecules obtained fromthese clones can be obtained directly from total DNA or from total RNA.The cDNA clones are not naturally occurring, but rather are preferablyobtained via manipulation of a partially purified naturally occurringsubstance (messenger RNA). The construction of a cDNA library from mRNAinvolves the creation of a synthetic substance (cDNA) and pureindividual cDNA clones can be isolated from the synthetic library byclonal selection of the cells carrying the cDNA library. Thus, theprocess which includes the construction of a cDNA library from mRNA andisolation of distinct cDNA clones yields an approximately 10⁶-foldpurification of the native message. Thus, purification of at least oneorder of magnitude, preferably two or three orders, and more preferablyfour or five orders of magnitude is expressly contemplated.

In another preferred embodiment, the nucleic acid molecules of theinvention comprise nucleotide sequences that (a) have the nucleic acidsequences set forth in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ IDNO:4, SEQ ID NO:5, SEQ ID NO:6, or a functional derivative thereof, orencode polypeptides having the full-length amino acid sequences setforth in SEQ ID NO:7, SEQ ID NO:8; SEQ ID NO:9, SEQ ID NO:10, SEQ IDNO:11, SEQ ID NO:12, or a functional derivative thereof; (b) is thecomplement of the nucleotide sequence of (a); (c) hybridizes underhighly stringent conditions to the nucleotide molecules of (a) andencodes a naturally occurring hOAT polypeptide; (d) encodes an hOATpolypeptide having the full-length amino acid sequence of the sequenceset forth in SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ IDNO:11, or SEQ ID NO:12 except that it lacks one or more of the domainsselected from the group consisting of an extracellular domain, atransmembrane domain, and an intracellular domain; or (e) is thecomplement of the nucleotide sequence of (d).

The nucleic acid molecules of the invention are isolated, enriched, orpurified preferably from a mammal, more preferably from a human.

Hybridization techniques may be used to isolate a nucleic acid moleculeof interest. Various low or high stringency hybridization conditions maybe used depending upon the specificity and selectivity desired.Stringency is controlled by varying salt or denaturant concentrations.Highly stringent conditions may mean conditions that are at least asstringent as the following: hybridization in 50% formamide, 5×SSC, 50 mMNaH₃PO₄, pH 6.8, 0.5% SDS, 0.1 mg/mL sonicated salmon sperm DNA, and 5×Denhart solution at 42° C. overnight; washing with 2×SSC, 0.1% SDS at45° C.; and washing with 0.2×SSC, 0.1% SDS at 45° C. Those skilled inthe art will recognize how such conditions can be varied to varyspecificity and selectivity.

Particularly preferred embodiments of this aspect of the invention arenaturally or non-naturally occurring variants of the nucleic acids ofthe invention. Among variants in this regard are variants that differfrom the aforementioned nucleic acid molecules by nucleotidesubstitutions, deletions or additions. The substitutions, deletions oradditions may involve one or more nucleotides. The variants may bealtered in coding or non-coding regions or both. Alterations in thecoding regions may produce conservative or non-conservative amino acidsubstitutions, deletions or additions. Such nucleic acid molecules areidentifiable as being able to hybridize to or which are at least about60-65% percent identical, preferably at least about 70-75% percentidentical, more preferably at least about 80-83% percent identical, andeven more preferably at least about 87-95% percent identical to thenucleic acid molecules shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3,SEQ ID NO:4, SEQ ID NO:5 or SEQ ID NO:6.

Among the particularly preferred embodiments of the invention in thisregard are nucleic acid molecules encoding polypeptides having the aminoacid sequences set forth in SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQID NO:10, SEQ ID NO:11, SEQ ID NO:12; variants, analogs, derivatives andfragments thereof, and fragments of the variants, analogs andderivatives.

Further particularly preferred in this regard are nucleic acid moleculesencoding OAT variants, analogs, derivatives and fragments, and variants,analogs and derivatives of the fragments, which have the amino acidsequence for the hOAT polypeptides of SEQ ID NO:7, SEQ ID NO:8, SEQ IDNO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12 in which several, a few,5 to 10, 1 to 5, 1 to 3, 2, 1 or no amino acid residues are substituted,deleted or added, in any combination. Especially preferred among theseare silent substitutions, additions and deletions, which do not alterthe properties and activities of the OAT polypeptides. Also especiallypreferred in this regard are conservative substitutions. In oneembodiment, the polypeptide encoded by the nucleic acid molecule is atleast about 30-35%, preferably at least about 40-45%, more preferably atleast about 50-55%, even more preferably at least about 60-65%, yet morepreferably at least about 70-75%, still more preferably at least about80-85%, and most preferably at least about 90-95% or more identical tothe amino acid sequence of SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ IDNO:10, SEQ ID NO:11, or SEQ ID NO:12.

In still another embodiment, the nucleic acid molecule encodes anaturally occurring variant of the polypeptide of SEQ ID NO:7, SEQ IDNO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, or SEQ ID NO:12 andhybridizes under stringent conditions to a nucleic acid moleculecomprising SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ IDNO:5 or SEQ ID NO:6, respectively.

Further preferred embodiments are variaSFnts of the herein describednucleic acid molecules in which the transporting activity of thepolypeptides is altered. Such alterations can result in eitherincreased, or decreased transport of substrates recognized by hOATpolypeptides. Further, variants can be made that result in OATpolypeptides which are no longer responsive to cellular signals, forexample, protein kinase C deactivation of transport.

Especially preferred are nucleic acid molecules encoding OATs which areexpressed on the basolateral (sinusoid) membrane of hepatocyte cells.

Included in the invention are all possible nucleic acid sequences thatencode the amino acid sequences of the hOAT polypeptides of theinvention. As it is recognized that alternate codons will encode thesame amino acid for most amino acids due to the degeneracy of thegenetic code, the sequences of this aspect includes nucleic acidsequences utilizing such alternate codon usage for one or more codons ofa coding sequence. For example, all four nucleic acid sequences GCT,GCC, GCA, and GCG encode the amino acid, alanine. Therefore, if for anamino acid there exists an average of three codons, a polypeptide of 100amino acids in length will, on average, be encoded by 3¹⁰⁰, or 5×10⁴⁷,nucleic acid sequences. Thus, a nucleic acid sequence can be modified(e.g., a nucleic acid sequence from an hOAT as specified above) to forma second nucleic acid sequence encoding the same polypeptide as encodedby the first nucleic acid sequence using routine procedures and withoutundue experimentation. Thus, all possible nucleic acid sequences thatencode the amino acid sequences of the hOAT polypeptides of theinvention are included in the present invention and are described, as ifall were written out in full, taking into account the codon usage.

The alternate codon descriptions are available in common textbooks, forexample, Stryer, BIOCHEMISTRY 3^(rd) ed., and Lehninger, BIOCHEMISTRY3^(rd) ed. Codon preference tables for various types of organisms areavailable in the literature. Because of the number of sequencevariations involving alternate codon usage, for the sake of brevity,individual sequences are not separately listed herein. Instead thealternate sequences are described by reference to the natural sequencewith replacement of one or more (up to all) of the degenerate codonswith alternate codons from the alternate codon table, preferably withselection according to preferred codon usage for the normal hostorganism or a host organism in which a sequence is intended to beexpressed. Those skilled in the art also understand how to alter thealternate codons to be used for expression in organisms where certaincodons code differently than shown in the “universal” codon table.

In a second aspect, the invention relates to a nucleic acid vectorcomprising a nucleic acid molecule encoding an hOAT polypeptide and apromoter element effective to initiate transcription in a host cell.

Those skilled in the art would recognize that a nucleic acid vector cancontain many other nucleic acid elements besides the promoter elementand the hOAT nucleic acid molecule. These other nucleic acid elementsinclude, but are not limited to, origins of replication, ribosomalbinding sites, nucleic acid sequences encoding drug resistance enzymesor amino acid metabolic enzymes, and nucleic acid sequences encodingsecretion signals, periplasm or peroxisome localization signals, orsignals useful for polypeptide purification.

A circular double stranded nucleic acid molecule can be cut and therebylinearized upon treatment with restriction enzymes. An assortment ofvectors, restriction enzymes, and the knowledge of the nucleotidesequences that the restriction enzymes operate upon are readilyavailable to those skilled in the art. A nucleic acid molecule of theinvention can be inserted into a vector by cutting the vector withrestriction enzymes and ligating the two pieces together.

Many techniques are available to those skilled in the art to facilitatetransformation or transfection of the expression construct into aprokaryotic or eukaryotic organism. These methods involve a variety oftechniques, such as treating the cells with high concentrations of salt,an electric field, or detergent, to render the host cell outer membraneor wall permeable to nucleic acid molecules of interest.

In a third aspect, the invention features a nucleic acid probe for thedetection of a nucleic acid encoding an hOAT polypeptide, fragment oranalogue in a sample. The nucleic acid probe contains nucleotides thatwill hybridize specifically to a sequence of at least 14 contiguousnucleotides set forth in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ IDNO:4, SEQ ID NO:5, or SEQ ID NO:6, or a functional derivative thereof.The probe is preferably at least 14 or more bases in length and selectedto hybridize specifically to a unique region of an hOAT encoding nucleicacid.

In preferred embodiments, the nucleic acid probe hybridizes to at least14 nucleotides of a nucleic acid encoding the full-length sequence setforth in SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ IDNO:11, SEQ ID NO:12, or a functional derivative thereof. Various low orhigh stringency hybridization conditions may be used depending upon thespecificity and selectivity desired. Under highly stringenthybridization conditions, only highly complementary nucleic acidsequences hybridize. Preferably, such conditions prevent hybridizationof nucleic acids having 1 or 2 mismatches out of 20 contiguousnucleotides.

In another preferred embodiment, the nucleic acid is an isolatedconserved or unique region, for example, those useful for the design ofhybridization probes to facilitate identification and cloning ofadditional polypeptides, or for the design of PCR probes to facilitateamplification or cloning of additional polypeptides. Such a nucleic acidmay contain additional sequences in addition to the conserved or uniqueregion, preferably 10-20 additional nucleotides on each side of theconserved or unique region, more preferably 30-40 additionalnucleotides, and most preferably 75-100 additional nucleotides.

Methods for using the probes include detecting the presence or amount ofhOAT polypeptide RNA in a sample by contacting the sample with a nucleicacid probe under conditions such that hybridization occurs and detectingthe presence or amount of the probe bound to hOAT polypeptide RNA. Thenucleic acid duplex formed between the probe and a nucleic acid sequencecoding for an hOAT polypeptide may be used in the identification of thesequence of the nucleic acid detected (for example see, Nelson et al.,in Nonisotopic DNA Probe Techniques, p. 275 Academic Press, San Diego(Kricka, ed., 1992) hereby incorporated by reference herein in itsentirety, including any drawings). Kits for performing such methods maybe constructed to include a container having disposed therein a nucleicacid probe.

Another feature of the invention is a nucleic acid molecule as set forthin SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, orSEQ ID NO:6, or fragments thereof, comprising one or more regions thatencode an hOAT polypeptide or an hOAT domain polypeptide, where the hOATpolypeptide or the hOAT domain polypeptide is fused to a non-hOATpolypeptide or amino-terminal tag. Possible non-hOAT polypeptide oramino-terminal tag fusion partners include, for example, but are notlimited to, glutathione-S-transferase (GST)-fusion proteins, greenfluorescent protein (GFP) and fusions with histidine residues asdescribed by Janknecht et al., 1991, Proc. Natl. Sci. USA 88:8972-8976.

The invention also features recombinant nucleic acid, preferably in acell or an organism. The recombinant nucleic acid may contain a sequenceset forth in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ IDNO:5, or SEQ ID NO:6 or a functional derivative thereof and a vector ora promoter effective to initiate transcription in a host cell. Therecombinant nucleic acid can alternatively contain a transcriptionalinitiation region functional in a cell, a sequence complimentary to anRNA sequence encoding an hOAT polypeptide and a transcriptionaltermination region functional in a cell.

Another aspect of the invention relates to a recombinant cell or tissuecomprising a nucleic acid molecule encoding an hOAT polypeptide or hOATpolypeptide fragment. The recombinant cell may comprise a nucleic acidmolecule encoding an hOAT polypeptide; an hOAT domain polypeptide; anhOAT polypeptide fragment or an hOAT polypeptide or polypeptide fragmentfused to a non-hOAT polypeptide. The recombinant cell can harbor anucleic acid vector that is extragenomic or intragenomic.

Extragenomic vectors are designed with their own origins of replicationallowing them to utilize the recombinant cell replication machinery tocopy and propagate the vector nucleic acid sequence. These vectors aresmall enough that they are not likely to harbor nucleic acid sequenceshomologous to genomic sequences of the recombinant cell.

Multiple intragenomic vectors are available to those skilled in the artand contain nucleic acid sequences that are homologous to nucleic acidsequences in a particular organism's genomic DNA. These homologoussequences will result in recombination events that integrate portions ofthe vector into the genomic DNA. Those skilled in the art can controlwhich nucleic acid sequences of the vector are integrated into the cellgenome by flanking the portion to be incorporated into the genome withhomologous sequences in the vector.

A new combination of genes or nucleic acid molecules can be introducedinto an organism using a wide array of nucleic acid manipulationtechniques available to those skilled in the art.

In yet another aspect, the invention features an isolated, enriched, orpurified hOAT polypeptide.

It is also advantageous for some purposes that an amino acid sequence bein purified form. Purification of at least one order of magnitude,preferably two or three orders, and more preferably four or five ordersof magnitude is expressly contemplated. The substance is preferably freeof contamination at a functionally significant level, for example 90%,95%, or 99% pure. The hOAT polypeptides, or fragments thereof, of thepresent invention are preferably isolated, purified, or enriched from amammal or a mammalian cell. The mammal is as defined herein andpreferably is a mouse, and most preferably is a human. Thesepolypeptides may be isolated, purified, or enriched from a cell thatcomprises an endogenous nucleic acid molecule that encodes thepolypeptide, or from a cell that is transformed with a nucleic acidmolecule that encodes the polypeptide. The polypeptide may also bechemically synthesized. Procedures for obtaining polypeptides using theabove methods are well known to those skilled in the art.

The polypeptides of the invention comprise an amino acid sequence having(a) the full-length amino acid sequence encoded by the nucleic acidsequences set forth in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ IDNO:4, SEQ ID NO:5, SEQ ID NO:6, or functional derivatives thereof, orthe amino acid sequences set forth in SEQ ID NO:7, SEQ ID NO:8; SEQ IDNO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, or functionalderivatives thereof.

The invention also features an isolated, enriched, or purified hOATpolypeptide fragment.

Examples of polypeptide fragments include hOAT domains, andhOAT-specific epitopes. Such fragments may, for example, be produced byproteolytic cleavage of the full-length protein. Preferably, thefragment is obtained recombinantly by appropriately modifying the DNAsequence encoding the proteins to delete one or more amino acids at oneor more sites of the C-terminus, N-terminus, and/or within the nativesequence. Fragments of a protein can be used as antigens for theproduction of hOAT-specific antibodies, or used as competitors ofsubstrates for hOAT polypeptides. It is understood that such fragmentsmay retain one or more characterizing portions of the native complex.Examples of such retained characteristics include: transportingactivity; substrate specificity; interaction with other molecules in theintact cell; regulatory functions; or binding with an antibody specificfor the native complex, or an epitope thereof.

Well-known examples of domains are the SH2 (Src Homology 2) domain(Sadowski, et al., (1986) Mol. Cell. Biol. 6:4396; Pawson andSchlessinger, (1993) Curr. Biol. 3:434), the SH3 domain (Mayer, et al.,(1988) Nature 332:272; Pawson and Schlessinger, (1993) Curr. Biol.3:434), and pleckstrin (PH) domain (Ponting, (1996) TIBS 21:245; Haslam,et al., (1993) Nature 363:309), all of which are domains that mediateprotein:protein interaction, and the kinase catalytic domain (Hanks andHunter, (1995) FASEB J 9:576-595). The relative homology is at least20%, more preferably at least 30% and most preferably at least 35%.Computer programs designed to detect such homologies are well known inthe art.

Comparisons between the sequences of two or more polynucleotides orpolypeptides can be performed using the local homology algorithm ofSmith and Waterman, (1981) Adv. Appl. Math. 2:482, by the homologyalignment algorithm of Needleman and Wunsch, (1970) J. Mol. Biol.48:443, or the method of Pearson and Lipman, (1988) PNAS 85:2444.Computer programs implementing these methods can be used and include,BLAST, GAP, BESTFIT, FASTA, and TFASTA which are offered in theWisconsin Genetics Software Package, Genetics Computer Group, 575Science Dr., Madison, Wis.

Protein motifs can be identified using computer programs readilyavailable to those skilled in the art. One such computer program isoffered by the Institute for Chemical Research at Kyoto University. TheKyoto computer program is offered at the following web site(http://www.motif.genome.ad.ip).

Protein topology, such as the orientation and location of transmembranehelixes can also be identified using readily available computerprograms. One such program is TopPred2 offered by Stockholm Universityand offered at the following web site(http://www.biokemi.su.se/˜server/toppred2/).

Another aspect of the invention features an isolated, enriched orpurified hOAT polypeptide analog.

The hOAT polypeptide analogs of the present invention preferably have asubstantially similar biological activity to the proteins encoded by thefull-length nucleic acid sequence set forth in SEQ ID NO:1, SEQ ID NO:2,SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, or SEQ ID NO:6, or the amino acidsequence set forth in SEQ ID NO:7, or SEQ ID NO:8; SEQ ID NO:9, SEQ IDNO:10, SEQ ID NO:11, or SEQ ID NO:12.

A sequence that is substantially similar will preferably have at least85% identity (more preferably at least 90% and most preferably 95-100%)to the amino acid sequence encoded by the nucleic acid sequence setforth in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ IDNO:5, or SEQ ID NO:6, or the amino acid sequence set forth in SEQ IDNO:7, SEQ ID NO:8; SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, or SEQ IDNO:12.

An hOAT polypeptide analog will retain some useful function such as, forexample, substrate binding, transport activity, or the ability to bindto an hOAT specific antibody (as defined below). The hOAT polypeptideanalog may be derived from a naturally occurring complex component byappropriately modifying the protein DNA coding sequence to add, remove,and/or to modify codons for one or more amino acids at one or more sitesof the C-terminus, N-terminus, and/or within the native sequence. It isunderstood that such analogs having added, substituted and/or additionalamino acids retain one or more characterizing portions of the nativehOAT polypeptides.

Examples of hOAT polypeptides analogs include, but are not limited to,(1) chimeric proteins which comprise a portion of an hOAT polypeptidesequence fused to a non-hOAT polypeptide sequence, for example, apolypeptide sequence of glutathione-S-transferase, (2) hOAT polypeptideslacking a specific domain, for example, the protein kinase C domain, (3)epitope-tagged hOAT polypeptides or fragments for immuno detection orpurification and (4) hOAT proteins having a point mutation.

Human OAT polypeptide analogs with deleted, inserted and/or substitutedamino acid residues may be prepared using standard techniques well knownto those of ordinary skill in the art. For example, the modifiedcomponents of the analogs may be produced using site-directedmutagenesis techniques (as exemplified by Adelman et al., (1983) DNA2:183) wherein nucleotides in the DNA coding the sequence are modifiedsuch that a modified coding sequence is produced, and thereafterexpressing this recombinant DNA in a prokaryotic or eukaryotic hostcell, using techniques such as those described above. Alternatively,proteins with amino acid deletions, insertions and/or substitutions maybe conveniently prepared by direct chemical synthesis, using methodswell known in the art. The analogs of the hOAT polypeptides may exhibitthe same qualitative biological activity as the hOAT polypeptidesthemselves.

In another aspect, the invention features an antibody (e.g., amonoclonal or polyclonal antibody) having specific-binding affinity toan hOAT polypeptide or hOAT polypeptide analog or fragment.

Antibodies having specific-binding affinity to an hOAT polypeptide maybe used in methods for detecting the presence and/or amount of an hOATpolypeptide in a sample by contacting the sample with the antibody underconditions such that an immunocomplex forms and detecting the presenceand/or amount of the antibody conjugated to the hOAT polypeptide.Diagnostic kits for performing such methods may be constructed toinclude a first container containing the antibody and a second containerhaving a conjugate of a binding partner of the antibody and a label,such as, for example, a radioisotope. The diagnostic kit may alsoinclude notification of an FDA approved use and instructions therefor.

For the production of polyclonal antibodies, various host animals may beimmunized by injection with the antigen. Various adjuvants may be usedto increase the immunological response, depending on the host species.

Monoclonal antibodies may be obtained by any technique which providesfor the production of antibody molecules by continuous cell lines inculture. Monoclonal antibodies may be obtained by methods known to thoseskilled in the art. See, for example, Kohler, et al., (1975) Nature256:495-497, and U.S. Pat. No. 4,376,110 issued Mar. 8, 1983, to Davidet al.

Still further, the invention features a hybridoma which produces anantibody having specific-binding affinity to an hOAT polypeptide.

In preferred embodiments, the hOAT antibody comprises a sequence ofamino acids that is able to specifically bind an hOAT polypeptide.

The invention features a method for identifying human cells containingan hOAT polypeptide or a related sequence. The method involvesidentifying the novel polypeptide in human cells using techniques thatare routine and standard in the art, such as those described herein foridentifying hOAT (e.g., cloning, Southern or Northern blot analysis,Western blot analysis, immunoassay, in situ hybridization, PCRamplification, etc.).

The invention also features methods of screening cells fornatural-binding partners of hOAT polypeptides. Binding partners includemodulators and downstream signaling molecules such as adaptor proteinsand may be identified by techniques well known in the art such asco-immunoprecipitation or by using, for example, a two-hybrid screen.(Fields and Song, U.S. Pat. No. 5,283,173, issued Feb. 1, 1994, andincorporated by reference herein). The present invention also featuresthe purified, isolated or enriched versions of the polypeptidesidentified by the methods described above.

The invention provides methods for screening compounds for their abilityto inhibit, or modulate the biological activity of the human organicanion transporter molecules of the invention. In preferred embodiments,cells expressing an hOAT polypeptide, including recombinant expressionconstructs of the invention, are contacted with a compound, andtransport activity is assayed. In especially preferred embodiments,competition assays, using known human organic anion transportersubstrates, such as p-aminohippurate, are used to test the ability of ascreened compound to interfere (compete) with the uptake of a knownsubstrate of an hOAT polypeptide. Additional preferred embodimentscomprise quantitative analyses of such effects.

The present invention is also useful for the detection of substrates,analogues of substrates, inhibitors and modulators, heretofore known orunknown, for the transporters of the invention, either naturallyoccurring or embodied as a drug. In preferred embodiments, suchsubstrates, analogues, or modulators may be detected in blood, saliva,semen, cerebrospinal fluid, plasma, lymph, vitreous humor, or any otherbodily fluid. In additional preferred embodiments, the inventionprovides methods for detecting and identifying substrates, analogues ofsubstrates, or modulators that preferentially affect either the uptakefunction or the efflux function of the transporters of the invention.

One method for identifying a substance capable of modulating an hOATpolypeptide activity comprises the steps of (a) contacting an hOATpolypeptide with a test substance; and (b) determining whether thesubstance alters the transporting activity of said polypeptide.

Another method of identifying substances capable of modulating thefunction of an hOAT polypeptide comprises the following steps: (a)expressing an hOAT polypeptide in cells; (b) adding a compound to thecells; and (c) monitoring the transporting activity of the hOAT.

A method of identifying substrates or inhibitors of hOAT polypeptidescomprises the following steps: (a) adding a test substrate to a cellline expressing an hOAT polypeptide; and (b) measuring the intracellularconcentration of the test substrate.

A second method of identifying substrates or inhibitors of hOATpolypeptides comprises the following steps: (a) adding a test substrateto a cell line expressing an hOAT polypeptide; (b) adding a substrateknown to be transported by said hOAT polypeptides; (c) measuring whethersaid test substrate competes with the uptake of the known substrate.

Another method of identifying substrates of an hOAT polypeptidecomprises the following steps: (a) attaching a fluorescent compound ontoa test compound; (b) adding said compound to a cell line expressing anhOAT polypeptide; and (c) monitoring whether said fluorescent compoundis taken into said cell line through the use of fluorescent microscopyor any other method which can detect fluorescence.

In a preferred embodiment, high-throughput screening employing 96-wellplates and a microtiter® fluorescence detection system is used to detectpotential modulators, substrates, analogues of substrates and inhibitorsof hOAT polypeptides which can compete with binding or transport offluorescent compounds known to bind or be transported by hOATpolypeptides.

In yet another embodiment, uptake of a test substrate by an hOATexpressing cell line is measured using knowledge of an hOAT's antiporteractivity. Uptake is measured by monitoring efflux of an accompanyingantiporter molecule. Efflux of the antiporter molecule can be detectedusing techniques known to those skilled in the art, for example,radiolabeling of the effluxed molecule and measurement using ascintillation counter. Measurement of antiporter efflux is also amenableto high-throughput screening.

A still further aspect of the invention is the identification ofsubstrates, analogues of substrates, inhibitors, or modulators thataffect the pharmacokinetics of drugs and compounds transported by hOATpolypeptides. Substances which decrease the transporting activity ofhOAT polypeptides are useful for increasing the half-life in the body ofdrugs and compounds excreted via human organic anion transporters.

Yet, another aspect of the invention involves substrates, andmodulators, which increase the transporting activity of an hOAT. Suchsubstances are advantageous for increasing the delivery of a drug orcompound, which is transported by an hOAT, to a target organ such askidney or liver. For example, a modulator which increases thetransporting activity of hOAT1 would increase the delivery of PAH orother drugs or compounds transported by hOAT1 to the kidney.

A last aspect of the invention features a cultured cell line whichstably expresses an hOAT polypeptide. Such cell lines are useful foridentifying substrates which are transported by hOAT polypeptides,inhibitors of hOAT polypeptides and for identifying modulators of hOATpolypeptides. Further, a panel of cells stably expressing hOATpolypeptides will be useful in studying the pharmacokinetics of variousdrugs and compounds. Preferably, the expression of an hOAT polypeptidein such cell lines is under the control of an inducible promoter. Morepreferably, said inducible promoter has a very low basal level ofexpression in such cell lines and high expression when induced.

A cell line stably expressing an hOAT polypeptide is useful fordesigning substrates which can be transported by an hOAT polypeptide ateither an increased or a decreased rate. Decreasing the rate oftransport can increase the effectiveness of some medicinals by makingtheir clearance from the body occur more slowly.

Cell lines stably expressing hOAT polypeptides are also useful for theidentification and development of drugs or compounds targeted to aparticular organ. For example, drugs or compounds identified assubstrates of hOAT1 would be good candidates for treating diseases ofthe kidneys whereas those identified as substrates of hOAT2 would begood candidates for treating diseases of the liver.

The OAT expressing cell lines are also useful for identifyingpotentially toxic compounds or drugs. For example, newly discovered ormodified drugs belonging to a class of drugs known to be toxic tocertain organs, e.g., kidney can be tested for transport against thepanel of cells expressing human OAT1 or hOAT3 polypeptides. If the newdrug or compound is transported by hOAT1 expressing cells, the new drugor compound may also be toxic to the kidney. However, if the new drug orcompound, belonging to a class known to be toxic, is not transported byhOAT1 or hOAT3 expressing cells than the new drug or compound would beexpected to be less toxic to the kidney than the parent class of drugsor compounds.

A preferred embodiment includes the identification of toxic compoundswhich are substrates of hOATs, and which can be used to treat diseasesof organs or tissues that express hOATs. For example, toxic compoundsfound to be transported by hOAT1 or hOAT3 would have potential use asanti-cancer drugs to treat cancers of the kidney.

The methods of the present invention can utilize any of the moleculesdisclosed in the invention. These molecules include nucleic acidmolecules encoding hOAT polypeptides, nucleic acid vectors, recombinantcells, polypeptides, or antibodies described herein.

The summary of the invention described above is non-limiting and otherfeatures and advantages of the invention will be apparent from thefollowing detailed description, and from the claims.

Definitions

By “isolated” in reference to nucleic acid it is meant a polymerpreferably consisting of 14 or more nucleotides conjugated to eachother, including DNA or RNA that is isolated from a natural source orthat is synthesized. The isolated nucleic acid of the present inventionis unique in the sense that it is not found in a pure or separated statein nature. Use of the term “isolated” indicates that a naturallyoccurring sequence has been removed from its normal cellular (i.e.,chromosomal) environment. Thus, the sequence may be in a cell-freesolution or placed in a different cellular environment. The term doesnot imply that the sequence is the only nucleotide sequence present, butthat it is essentially free (about 90-95% pure at least) of nucleotidematerial naturally associated with it and thus is meant to bedistinguished from isolated chromosomes.

By the use of the term “enriched” in reference to nucleic acid it ismeant that the specific DNA or RNA sequence constitutes a significantlyhigher fraction (2-5 fold) of the total DNA or RNA present in the cellsor solution of interest than in normal or diseased cells or in the cellsfrom which the sequence was taken. This could be caused by apreferential reduction in the amount of other DNA or RNA present, or bya preferential increase in the amount of the specific DNA or RNAsequence, or by a combination of the two. However, it should be notedthat “enriched” does not imply that there are no other DNA or RNAsequences present, just that the relative amount of the sequence ofinterest has been significantly increased.

The term “significant” here is used to indicate that the level ofincrease is useful to the person making such an increase, and generallymeans an increase relative to other nucleic acids of about at least 2fold, more preferably at least 5 to 10 fold or even more. The term alsodoes not imply that there is no DNA or RNA from other sources. The othersource DNA may, for example, comprise DNA from a yeast or bacterialgenome, or a cloning vector such as pUC19. This term distinguishes thesequence from naturally occurring enrichment events, such as viralinfection, or tumor type growths, in which the level of one mRNA may benaturally increased relative to other species of mRNA. That is, the termis meant to cover only those situations in which a person has intervenedto elevate the proportion of the desired nucleic acid.

The term “purified” in reference to nucleic acid does not requireabsolute purity (such as a homogeneous preparation); instead, itrepresents an indication that the sequence is relatively purer than inthe natural environment (compared to the natural level this level shouldbe at least 2-5 fold greater, e.g., in terms of mg/mL). The term is alsochosen to distinguish clones already in existence which may encode anhOAT polypeptide but which have not been isolated from other clones in alibrary of clones. Thus, the term covers clones encoding an hOATpolypeptide which are isolated from other non-hOAT clones.

The term “nucleic acid molecule” describes a polymer ofdeoxyribonucleotides (DNA) or ribonucleotides (RNA). The nucleic acidmolecule may be isolated from a natural source by cDNA cloning orsubtractive hybridization or synthesized manually. The nucleic acidmolecule may be synthesized manually by the triester synthetic method orby using an automated DNA synthesizer.

The term “cDNA cloning” refers to hybridizing a small nucleic acidmolecule, a probe, to cDNA. The probe hybridizes (binds) tocomplementary sequences of cDNA.

The term “complementary” describes two nucleotides that can formmultiple favorable interactions with one another. For example, adenineis complementary to thymine as they can form two hydrogen bonds.Similarly, guanine and cytosine are complementary since they can formthree hydrogen bonds. Thus if a nucleic acid sequence contains thefollowing sequence of bases, thymine, adenine, guanine and cytosine, a“complement” of this nucleic acid molecule would be a moleculecontaining adenine in the place of thymine, thymine in the place ofadenine, cytosine in the place of guanine, and guanine in the place ofcytosine. Because the complement can contain a nucleic acid sequencethat forms optimal interactions with the parent nucleic acid molecule,such a complement can bind with high affinity to its parent molecule.

The term “hybridize” refers to a method of interacting a nucleic acidsequence with a DNA or RNA molecule in solution or on a solid support,such as cellulose or nitrocellulose. If a nucleic acid sequence binds tothe DNA or RNA molecule with high affinity, it is said to “hybridize” tothe DNA or RNA molecule. The strength of the interaction between theprobing sequence and its target can be assessed by varying thestringency of the hybridization conditions. Under highly stringenthybridization conditions, only highly complementary nucleic acidsequences hybridize. Preferably, such conditions prevent hybridizationof nucleic acids having one or two mismatches out of 20 contiguousnucleotides.

By “conserved nucleic acid regions”, it is meant regions present on twoor more nucleic acids encoding an hOAT polypeptide, to which aparticular nucleic acid sequence can hybridize under lower stringencyconditions. Examples of lower stringency conditions suitable forscreening for nucleic acids encoding hOAT polypeptides are provided inAbe, et al. (1992) J. Biol. Chem. 19:13361 (hereby incorporated byreference herein in its entirety, including any drawings). Preferably,conserved regions differ by no more than 5 out of 20 contiguousnucleotides.

By “unique nucleic acid region” is meant a sequence present in afull-length nucleic acid coding for an hOAT polypeptide that is notpresent in a sequence coding for any other known naturally occurringpolypeptide. Such regions preferably comprise 12 or more contiguousnucleotides present in the full-length nucleic acid encoding an hOATpolypeptide. In particular, a unique nucleic acid region is preferablyof human origin.

The term “nucleic acid vector” relates to a single or double strandedcircular nucleic acid molecule that can be transfected or transformedinto cells and replicate independently or within the host cell genome.

The terms “transformation” and “transfection” refer to methods ofinserting an expression construct into a cellular organism.

The term “promoter element” describes a nucleotide sequence that isincorporated into a vector that, once inside an appropriate cell, canfacilitate transcription factor and/or polymerase binding and subsequenttranscription of portions of the vector DNA into mRNA. The promoterelement precedes the 5′ end of the hOAT polypeptide nucleic acidmolecule such that the latter is transcribed into mRNA. Host cellmachinery then translates mRNA into a polypeptide.

By an “hOAT polypeptide” is meant the full-length amino acid sequence ofSEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQID NO:12, or a functional derivative thereof.

The term “recombinant cell” refers to a cell that has been altered tocontain a new combination of genes or nucleic acid molecules.

The term “recombinant organism” refers to an organism that has a newcombination of genes or nucleic acid molecules.

The term “organism” relates to any living being comprised of at leastone cell. An organism can be as simple as one eukaryotic cell or ascomplex as a mammal. Therefore, a recombinant organism can also be arecombinant cell, which may be a eukaryotic or a prokaryotic organism.

By “recombinant hOAT polypeptide” it is meant to include a polypeptideproduced by recombinant DNA techniques such that it is distinct from anaturally occurring polypeptide either in its location (e.g., present ina different cell or tissue than found in nature), purity or structure.Generally, such a recombinant polypeptide will be present in a cell inan amount different from that normally observed in nature.

The term “eukaryote” refers to an organism comprised of cells thatcontain a nucleus. Eukaryotes are differentiated from “prokaryotes”which do not have a nucleus and lack other cellular structures found ineukaryotes, such as mitochondria and endoplasmic reticulum. Prokaryotesinclude unicellular organisms, such as bacteria, while eukaryotes arerepresented by yeast, invertebrates, and vertebrates.

The term “extragenomic” refers to a nucleic acid vector which does notinsert into the cell genome. Thus, these vectors replicate independentlyof the host genome and do not recombine with or integrate into thegenome.

The term “intragenomic” defines a nucleic acid construct that isincorporated within the cell genome.

By “isolated” in reference to a polypeptide is meant a polymer of 6, 12,18 or more amino acids conjugated to each other, including polypeptidesthat are isolated from a natural source or that are synthesized. Theisolated polypeptides of the present invention are unique in the sensethat they are not found in a pure or separated state in nature. Use ofthe term “isolated” indicates that a naturally occurring sequence hasbeen removed from its normal cellular environment. Thus, the sequencemay be in a cell-free solution or placed in a different cellularenvironment. The term does not imply that the sequence is the only aminoacid chain present, but that it is essentially free (about 90-95% pureat least) of material naturally associated with it.

By the use of the term “enriched” in reference to a polypeptide it ismeant that the specific amino acid sequence constitutes a significantlyhigher fraction (2-5 fold) of the total of amino acid sequences presentin the cells or solution of interest than in normal or diseased cells orin the cells from which the sequence was taken. This could be caused bya preferential reduction in the amount of other amino acid sequencespresent, or by a preferential increase in the amount of the specificamino acid sequence of interest, or by a combination of the two.However, it should be noted that “enriched” does not imply that thereare no other amino acid sequences present, just that the relative amountof the sequence of interest has been significantly increased.

The term “significant” here is used to indicate that the level ofincrease is useful to the person making such an increase, and generallymeans an increase relative to other amino acid sequences of about atleast 2 fold, more preferably at least 5 to 10 fold or even more. Theterm also does not imply that there are no amino acid sequences fromother sources. The other source amino acid sequences may, for example,comprise amino acid sequences encoded by a yeast or bacterial genome, ora cloning vector such as pUC19. The term is meant to cover only thosesituations in which a person has intervened to elevate the proportion ofthe desired amino acid sequences.

The term “purified” in reference to a polypeptide does not requireabsolute purity (such as a homogeneous preparation); instead, itrepresents an indication that the sequence is relatively purer than inthe natural environment (compared to the natural level this level shouldbe at least 2-5 fold greater, e.g., in terms of mg/mL).

By “an hOAT polypeptide fragment” it is meant an amino acid sequencethat is less than the full-length hOAT amino acid sequences (and whichexcludes the listed EST sequences) encoded by the nucleic acid sequenceset forth in SEQ ID NO:1 (excluding EST clone R25797), SEQ ID NO:2(excluding EST clone A1016020), SEQ ID NO:3 (excluding EST clone A1016020), SEQ ID NO:4 (excluding EST clone H41333), SEQ ID NO:5, or SEQID NO: 6 (excluding EST clone AA705512) or the amino acid sequence(excluding the amino acids encoded by the EST clones previously listedset forth in SEQ ID NO:7, SEQ ID NO:8; SEQ ID NO:9, SEQ ID NO:10, SEQ IDNO:11, or SEQ ID NO:12. Thus, the term “fragment” is used to indicate apolypeptide derived from the amino acid sequence of the hOATpolypeptides, of the complexes having a length less than the full-lengthpolypeptide from which it has been derived.

By “an hOAT domain” it is meant a portion of the hOAT polypeptide havinghomology to amino acid sequences from one or more known proteins whereinthe sequence predicts some common function, interaction or activity.

By “hOAT polypeptide analog” it is meant an amino acid sequencesubstantially similar to the sequence encoded by the nucleic acidsequence set forth in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ IDNO:4, SEQ ID NO:5. or SEQ ID NO:6, or the amino acid sequence set forthin SEQ ID NO:7, or SEQ ID NO:8; SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11,or SEQ ID NO:12.

By “biological activity” it is meant an activity of the hOATpolypeptides in a cell. The biological activity of the hOAT polypeptidesis related to some of the activities of the cell which include, but arenot limited to, transport (uptake or excretion of a substrate from acell), cell proliferation, mitogenesis, metastasis, tumor escape, celladhesion, transformation, or apoptosis.

By “identity” is meant a property of sequences that measures theirsimilarity or relationship. Identity is measured by dividing the numberof identical residues in the two sequences by the total number ofresidues and multiplying the product by 100. Thus, two copies of exactlythe same sequence have 100% identity, but sequences that are less highlyconserved and have deletions, additions, or replacements have a lowerdegree of identity. Those skilled in the art will recognize that severalcomputer programs are available for determining sequence identity.

An hOAT polypeptide analog may differ from the native sequence of anhOAT polypeptide in that one or more amino acids have been changed,added or deleted. Changes in amino acids may be conservative ornon-conservative. By “conservative” it is meant the substitution of anamino acid for one with similar properties such as charge,hydrophobicity, structure, etc.

By “hOAT-specific epitope” it is meant a sequence of amino acids that isboth antigenic and unique to hOAT.

By “specific-binding affinity” is meant that the antibody binds totarget (hOAT) polypeptides with greater affinity than it binds to otherpolypeptides under specified conditions.

The term “polyclonal” refers to antibodies that are heterogenouspopulations of antibody molecules derived from the sera of animalsimmunized with an antigen or an antigenic functional derivative thereof.

“Monoclonal antibodies” are substantially homogenous populations ofantibodies to a particular antigen.

The term “antibody fragment” refers to a portion of an antibody, oftenthe hypervariable region and portions of the surrounding heavy and lightchains, that displays specific binding affinity for a particularmolecule. A hypervariable region is a portion of an antibody thatphysically binds to the polypeptide target.

By “hybridoma” is meant an immortalized cell line which is capable ofsecreting an antibody, for example an hOAT antibody.

By “natural-binding partner” it is meant a protein, organic anion, orother molecule that interacts with an hOAT polypeptide.

As used herein, pharmacokinetics is the process by which a drug orcompound is absorbed, distributed, metabolized and eliminated by thebody.

The term “compound” includes small organic or inorganic molecules ofmolecular weight of preferably less than 1000 atomic units, morepreferably less than 800 atomic units, and most preferably less than 500atomic units. “Organic molecules” include all molecules that contain acarbon atom, whereas “inorganic molecules” are those that do not have acarbon atom.

The term “function” refers to the cellular role of a protein. The roleof the proteins of the invention may include transport of substratesinto and out of a cell, involvement in cascades controlling cell growth,migration, differentiation, gene expression, muscle contraction, glucosemetabolism, cellular protein synthesis, and regulation of the cellcycle.

The term “modulates” refers to the ability of a compound to alter thefunction of an hOAT polypeptide. A modulator preferably activates ordecreases the transporter activity of an hOAT protein. A modulator thatincreases the transporting activity is a positive modulator; and onethat decreases the transporting activity is a negative modulator. Theterm “modulates” also refers to altering the function of a protein byincreasing or decreasing the probability that a complex, i.e. anassembly of at least two molecules bound to one another, forms betweenan hOAT protein and a natural-binding partner.

The term “transporting activity,” in the context of the invention,defines the ability of a transporter polypeptide to uptake a substrateinto a cell or efflux a molecule out of a cell.

The term “substrate” as used herein refers to a molecule that istransported into or out of a cell by an OAT polypeptide. The substratemay be an organic compound or molecule, inorganic compound or molecule,a peptide, or a protein.

The term “activates” refers to increasing the transport or efflux of amolecule into or out of a cell.

The term “inhibitor” refers to a compound or substance that binds to asubstrate-binding site and, thereby, decreases or prevents transport ofan hOAT substrate.

The term “expressing” as used herein refers to the production of an hOATpolypeptide from a nucleic acid vector containing an hOAT gene within acell.

The term “adding” as used herein refers to administering a solutioncomprising a compound to the medium bathing cells.

The term “functional derivative” with respect to a polypeptide is apolypeptide that possesses a biological activity (either functional orstructural) or an immunological characteristic that is substantiallysimilar to a biological activity or an immunological characteristic of anon-recombinant hOAT. A functional derivative of an hOAT polypeptide mayor may not contain post-translational modifications such as covalentlylinked carbohydrate, depending on the necessity of such modificationsfor the performance of a specific function. The term “functionalderivative” is intended to include the “fragments”, “variants”,“analogues”, “homologues” or chemical derivatives of a molecule.

Similarly, a “functional derivative” of a gene encoding an hOATpolypeptide of the present invention includes “fragments”, variants”, or“analogues” of the gene, which may be “substantially similar” innucleotide sequence, and which encode a molecule possessing similaractivity to an hOAT polypeptide or fragment thereof. Permutationsresulting from degeneracy of the genetic code are also consideredfunctional derivatives.

A molecule is said to be “substantially similar” to another molecule ifthe sequence of amino acids in both molecules is substantially the same.Substantially similar amino acid molecules will possess a similarbiological activity. Thus, provided that two molecules possess a similaractivity, they are considered variants as that term is used herein evenif one of the molecules contains additional amino acid residues notfound in the other, or if the sequence of amino acid residues is notidentical.

A “chemical derivative” of a polypeptide contains additional chemicalmoieties not normally a part of the polypeptide.

The term “mammalian” refers to such organisms as mice, rats, rabbits,goats, more preferably monkeys and apes, and most preferably humans.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the cDNA and deduced amino acid sequences of human OAT1.

FIG. 2 shows the cDNA and deduced amino acid sequences of human OAT2A.

FIG. 3 shows the cDNA and deduced amino acid sequences of human OAT2B.

FIG. 4 shows the cDNA and deduced amino acid sequences of human OAT3.

FIG. 5 shows the cDNA and deduced amino acid sequences of human OAT4.

FIG. 6 shows the cDNA and deduced amino acid sequences of human OAT5.

FIG. 7 shows the amount of ³H-PAH taken up by HeLa cells transfectedwith increasing amounts of hOAT1 or vector control (LacZ) cDNAs.

FIG. 8 shows that oocytes injected with hOAT1 RNA uptake ³H-PAH to amuch greater extent than water injected oocytes.

FIG. 9 shows that ECR293 cells stably containing hOAT1, in plasmidpIND(sp1), uptake approximately 50-fold more ³H-PAH when induced byponasterone than non-induced cells.

FIG. 10 shows that ECR293 cells stably containing hOAT2A, in plasmidpIND(sp1), uptake approximately 2.6-fold more ³H-PAH when induced byponasterone than non-induced cells.

FIG. 11 shows that sulfobromophthalein but not α-ketoglutarate competedwith ³H-PAH uptake in ECR293/OAT2A cells induced with ponasterone.

FIG. 12 shows that oocytes injected with hOAT3 RNA uptake ³H-PAH to amuch greater extent than water injected oocytes.

FIG. 13 (A-E) shows Northern Blot analyses of hOAT1-5 mRNA in varioushuman tissues.

DETAILED DESCRIPTION OF THE INVENTION

A. Introduction

Organic anions are carbon-based molecules with one or more negativecharges at physiological pH. They can be endogenous compounds such asbile acids or xenobiotics such as p-aminohippurate. Organic aniontransporters are membrane proteins that facilitate the transport oforganic anions across the cell membrane. The family described hereinconsists of proteins sharing at least 50% amino acid sequence similarityamong each other. They show broad substrate specificity, meaning thateach member can transport a variety of organic anions. Hence, they areoften referred to as multi-specific organic anion transporters. Themechanism of transport, though not currently elucidated, probably is asecondary or tertiary active transport involving exchange of anotherorganic anion. An example of a tertiary transport system is the ratkidney OAT1 transporter. The primary transport system is theNa⁺/K⁺-ATPase, which establishes the high out-to-in Na⁺ gradient. ThisNa⁺ gradient then drives the cotransport of Na⁺ and dicarboxylates suchas α-ketoglutarate through a secondary Na⁺/dicarboxylate transporter.Finally, the tertiary transport system is established by OAT1, whichexchanges external PAH for intracellular α-ketoglutarate.

The liver and kidney are two organs rich in organic anion transporters.Hepatocytes and epithelial cells of the kidney proximal tubules arepolarized cells with a basolateral and apical membrane. For thehepatocyte, the basolateral membrane faces the blood-filled sinusoid.Thus, the basolateral membrane is also called the sinusoidal membrane.The apical membrane faces the canaliculus, into which bile is secreted.The hepatocyte thus can transport organic anions from the blood into thebile canaliculus across the basolateral and apical membranes. Similarly,cells of the kidney proximal tubule transport organic anions from theblood into urine across their basolateral and apical membranes. Examplesof liver sinusoidal transporters are the oatp1 and oatp2, whereas OAT1is a kidney basolateral transporter.

B. General Method

The practice of the present invention employs conventional techniques ofmolecular and cell biology, microbiology, and recombinant DNA. Mosttechniques are well-described in J. Sambrook et al., “Molecular Cloning;A Laboratory Manual” (1989) and J. Celis, ed. “Cell Biology: ALaboratory Handbook” (1998).

Identification of hOAT Nucleic Acid Molecules

Extensive searches of Genbank databases were performed using databasetools available through the website maintained by the National Centerfor Biotechnology Information (http://www.ncbi.nlm.nih.gov/). Inparticular, the Basic Local Alignment Search Tool (BLAST) was used toperform sequence similarity searches of the Genbank databases, such asthe database containing human expressed sequence tags (ESTs). The ESTdatabase contains sequences of randomly selected clones from a varietyof human cDNA libraries. Since each EST clone was sequenced with oneprimer, about 300-500 bp of sequence information is available from eachclone. The BLAST programs can translate these sequences in all possiblereading frames and compare them to an input protein sequence. Human ESTsshowing homology to rat OAT proteins where hereby identified.

Identified EST clones can be obtained from licensed distributors of theI.M.A.G.E. Consortium Lawrence Livermore National Laboratory (LLNL) cDNAclones. The clones are then sequenced to verify their homologies topublished sequences. DNA sequencing is usually done by Sanger's methodof using dideoxynucleotide triphosphates in a primer extension reaction.The DNA to be sequenced, called the template, is first denatured andthen annealed to a specific primer, usually between 15-25 basepairs. Inone method, ³⁵S-adenosine triphosphate is incorporated into the growingDNA strand by a DNA polymerase. Materials for such a reaction areavailable from commercial kits such as Sequenase 2.0® from Amersham.Reaction products are separated on a long polyacrylamide gel, usually6.0%, by electrophoresis using Tris-borate buffer. Afterelectrophoresis, the gel is dried under vacuum and then exposed toautoradiographic film. The DNA sequence can be read from the autoradimage and entered into computer for sequence analysis. Alternatively,fluorescent-tagged nucleotides can be incorporated by the polymerasechain reaction (PCR) into the template DNA using the thermal-stablepolymerase from Thermus aquaticus (Taq polymerase). The PCR products canthen be loaded onto an automated sequencer such as the ABI 377® (PEApplied Biosystems, Foster City, Calif.), which separates the productsand determines the sequence.

Isolation of cDNA clones from a particular tissue requires theconstruction of a cDNA library representing messages of that tissue.Library construction entails the isolation of mRNA from the sourcetissue and usually a fractionation step to separate low (<0.5 kb) andhigh molecular weight species. The mRNAs are then primed with a DNAoligonucleotide, either a poly-thymidine or a mixture of random primers.Reverse transcriptase is then added to synthesize the complementary DNAstrand. Short DNA fragments with sticky ends, called adapters, areligated to the cDNAs so they can be subcloned into an appropriatevector. Popular vectors include bacteriophages such as λgt11 or λZAP(Stratagene, La Jolla, Calif.). For phage libraries, the cDNAs areligated to lambda phage arms at the adapter site and then packaged intophage particles. Once packaged, the library can then be amplified byinfecting an appropriate bacterial host. Ready-made libraries of varioushuman tissues are available from several commercial sources.

To isolate a particular gene of interest from the cDNA library, analiquot is plated out onto large plates and then transferred tonitrocellulose or nylon membrane. The phage DNA attached to the membraneis denatured in alkali solution, neutralized, and then crosslinked tothe filter by baking or UV irradiation. The filter is then hybridizedwith a probe of interest. The probe can be an oligonucleotide or alonger, double-stranded fragment of DNA, such as the insert of an ESTclone. The double-stranded probe is usually labeled with ³²P to highspecific activity (>10⁹ dpm/μg), denatured by boiling for 2 minutes, andadded to a hybridization buffer to concentration of 10⁶ dpm/ml.Hybridization buffers can vary in composition depending on thehybridization condition desired. One common formulation contains 0.9 MNaCl, 90 mM sodium citrate (pH 7.0), 10 mM EDTA (pH 8.0), 0.1% (w/v)Ficoll, 0.1% (w/v) polyvinylpyrrolidone, 0.1 (w/v) BSA, 0.5% SDS, and100 μg/ml sheared, denatured salmon sperm DNA. Other formulations areavailable from commercial sources. The filters are hybridized with theprobe at 65° C. overnight or at least 16 hours. They are then washedwith sodium citrate solutions with decreasing ionic strength andincreasing temperature. Finally, they are exposed to autoradiographicfilm with an intensifying screen at −80° C. overnight.

If a plaque contains DNA sequences complementary to the probe, the probewill hybridize to the complementary sequences on the filter. The plaquecontaining the complementary sequences is detected by exposing X-rayfilm to the filter. The plaque of interest can then be isolated andpurified by conventional methods. Furthermore, once purified, the phageDNA can be manipulated so the relevant cDNA insert is excised from thephage into a phagmid by an in vivo excision procedure using appropriatehost cells. The phagmids can then be amplified in bacteria and isolatedby conventional methods such as alkaline lysis. Once the plasmid ispurified, it can be analyzed by standard means such as restrictionendonuclease or PCR mapping and DNA sequencing. DNA sequence informationis analyzed for open reading frames and compared to published sequences.As discussed earlier, sets of computer programs for DNA and proteinsequence analysis are commercially available.

Once a cDNA coding for a full-length transporter protein is isolated, itcan be excised from its cloning vector and subcloned into an appropriateexpression vector. Subcloning is a frequently used procedure inrecombinant DNA technology. It involves the digestion of DNA byrestriction endonucleases, fractionation of the DNA fragments by agarosegel electrophoresis, and purification of the desired fragment by aDNA-binding matrix. The purified fragment is then ligated to a precutvector of choice by adding an enzyme such as T4 DNA ligase. The new DNAconstruct can then be transfected into a mammalian cell line to test forfunctional expression. Many types of expression plasmids arecommercially available and they all share common elements. An expressionplasmid usually contains a bacterial origin of replication, a DNApromoter that is active in a mammalian cell line, a multiple cloningsite, a transcriptional termination element, and a selective marker thatconfers resistance to an antibiotic. An expression plasmid harboring thecDNA of interest can be transfected into mammalian cells by severalconventional means like calcium phosphate precipitation or lipofection.Many transfection kits are commercially available.

Recombinant Cells Containing a Nucleic Acid Molecule Encoding an hOAT

Cell lines which stably express hOAT polypeptides can be isolated. Suchcells may be obtained by using expression vectors which carry nucleicacid sequences which encode hOAT polypeptides. The expression vectorsmay contain viral origins of replication and/or endogenous expressionelements. Further, such expression vectors may carry nucleic acidsequences which encode for a selectable marker.

A nucleic acid molecule, such as DNA, is said to be “capable ofexpressing” a polypeptide if it contains nucleotide sequences whichcontain transcriptional and translational regulatory information andsuch sequences are “operably linked” to nucleotide sequences whichencode the polypeptide. An operable linkage is a linkage in which theregulatory DNA sequences and the DNA sequence sought to be expressed areconnected in such a way as to permit gene sequence expression. Theprecise nature of the regulatory regions needed for gene sequenceexpression may vary from organism to organism, but shall in generalinclude a promoter region which, in prokaryotes, contains both thepromoter (which directs the initiation of RNA transcription) as well asthe DNA sequences which, when transcribed into RNA, will signalsynthesis initiation. Such regions will normally include those5′-non-coding sequences involved with initiation of transcription andtranslation, such as the TATA box, capping sequence, CAAT sequence, andthe like.

If desired, the non-coding region 3′ to the sequence encoding an hOATpolypeptide of the invention may be retained for its transcriptionaltermination regulatory sequences, such as termination andpolyadenylation. Thus, by retaining the 3′-region naturally contiguousto the DNA sequence encoding an hOAT polypeptide of the invention, thetranscriptional termination signals may be provided. Where thetranscriptional termination signals are not satisfactorily functional inthe expression host cell, then a 3′ region functional in the host cellmay be substituted.

Two DNA sequences (such as a promoter region sequence and a sequenceencoding an hOAT polypeptide of the invention) are said to be operablylinked if the nature of the linkage between the two DNA sequences doesnot (1) result in the introduction of a frame-shift mutation, (2)interfere with the ability of the promoter region sequence to direct thetranscription of a gene sequence encoding an hOAT polypeptide of theinvention, or (3) interfere with the ability of the gene sequence of anhOAT polypeptide of the invention to be transcribed by the promoterregion sequence. Thus, a promoter region would be operably linked to aDNA sequence if the promoter were capable of effecting transcription ofthat DNA sequence. Thus, to express a gene encoding an hOAT polypeptideof the invention, transcriptional and translational signals recognizedby an appropriate host are necessary.

The present invention encompasses the expression of a gene encoding anhOAT polypeptide of the invention (or a functional derivative thereof)in either prokaryotic or eukaryotic cells. Prokaryotic hosts are,generally, very efficient and convenient for the production ofrecombinant proteins and for the propagation of clones. Prokaryotes mostfrequently are represented by various strains of E. coli. However, othermicrobial strains may also be used, including other bacterial strains.

In prokaryotic systems, plasmid vectors that contain replication sitesand control sequences derived from a species compatible with the hostmay be used. Examples of suitable plasmid vectors may include pBR322,pUC118, pUC119 and the like; suitable phage or bacteriophage vectors mayinclude % λt10, λgt11 and the like; and suitable virus vectors mayinclude pMAM-neo, pKRC and the like. Preferably, the selected vector ofthe present invention has the capacity to replicate in the selected hostcell.

Recognized prokaryotic hosts include bacteria such as E. coli, Bacillus,Streptomyces, Pseudomonas, Salmonella, Serratia, and the like. However,under such conditions, the polypeptide will not be glycosylated. Theprokaryotic host must be compatible with the replicon and controlsequences in the expression plasmid.

To express an hOAT polypeptide of the invention (or a functionalderivative thereof) in a prokaryotic cell, it is necessary to operablylink the sequence encoding the hOAT polypeptide of the invention to afunctional prokaryotic promoter. Such promoters may be eitherconstitutive or, more preferably, regulatable (i.e., inducible orderepressible). Examples of constitutive promoters include the intpromoter of bacteriophage λ, the bla promoter of the β-lactamase genesequence of pBR322, and the cat promoter of the chloramphenicol acetyltransferase gene sequence of pPR325, and the like. Examples of inducibleprokaryotic promoters include the major right and left promoters ofbacteriophage λ (P_(L) and P_(R)), the trp, recA, λacZ λacI, and galpromoters of E. coli, the α-amylase (Ulmanen et al., (1985) J.Bacteriol. 162:176-182), and the promoters of the bacteriophages ofBacillus (Gryczan, In: The Molecular Biology of the Bacilli, AcademicPress, Inc., NY, 1982), and Streptomyces promoters (Ward et al., (1986)Mol. Gen. Genet. 203:468-478). Prokaryotic promoters are reviewed byGlick (1987, Ind. Microbiot. 1:277-282), Cenatiempo (1986, Biochimie68:505-516), and Gottesman (1984, Ann. Rev. Genet. 18:415-442).

Proper expression in a prokaryotic cell also requires the presence of aribosome-binding site upstream of the gene sequence-encoding sequence.Such ribosome-binding sites are disclosed, for example, by Gold et al.(1981, Ann. Rev. Microbiol. 35:365-404). The selection of controlsequences, expression vectors, transformation methods, and the like, aredependent on the type of host cell used to express the gene. As usedherein, “cell”, “cell line”, and “cell culture” may be usedinterchangeably and all such designations include progeny. Thus, thewords “transformants” or “transformed cells” include the primary subjectcell and cultures derived therefrom, without regard to the number oftransfers. It is also understood that all progeny may not be preciselyidentical in DNA content, due to deliberate or inadvertent mutations.However, as defined, mutant progeny have the same functionality as thatof the originally transformed cell.

Host cells which may be used in the expression systems of the presentinvention are not strictly limited, provided that they are suitable foruse in the expression of the hOAT polypeptide of interest. Especiallysuitable hosts for expressing the polypeptides of the invention areeukaryotic cells. Preferred eukaryotic hosts include, for example,yeast, fungi, insect cells, amphibian oocytes, mammalian cells either invivo, or in tissue culture. Mammalian cells which may be useful as hostsinclude 3T3-L1 preadipocytes, cells of fibroblast origin such as VERO orCHO-K1, or cells of lymphoid origin and their derivatives. Preferredmammalian host cells include HeLa, EcR293, SP2/0 and J558L, as well asneuroblastoma cell lines such as IMR 332, which may provide bettercapacities for correct post-translational processing.

In addition, plant cells are also available as hosts, and controlsequences compatible with plant cells are available, such as thecauliflower mosaic virus 35S and 19S, and nopaline synthase promoter andpolyadenylation signal sequences. Another preferred host is an insectcell, for example the Drosophila larvae. Using insect cells as hosts,the Drosophila alcohol dehydrogenase promoter can be used (Rubin, (1988)Science 240:1453-1459). Alternatively, baculovirus vectors can beengineered to express large amounts of hOAT polypeptides of theinvention in insect cells (Jasny, (1987) Science 238:1653; Miller etal., In: Genetic Engineering, Vol. 8, Plenum, Setlow et al., eds., pp.277-297, 1986).

Any of a series of yeast expression systems can be utilized whichincorporate promoter and termination elements from the activelyexpressed sequences coding for glycolytic enzymes that are produced inlarge quantities when yeast are grown in mediums rich in glucose. Knownglycolytic gene sequences can also provide very efficienttranscriptional control signals. Yeast provides substantial advantagesin that it can also carry out post-translational modifications. A numberof recombinant DNA strategies exist utilizing strong promoter sequencesand high copy number plasmids which can be utilized for production ofthe desired proteins in yeast. Yeast recognizes leader sequences oncloned mammalian genes and secretes peptides bearing leader sequences(i.e., pre-peptides). Several possible vector systems are available forthe expression of hOAT polypeptides of the invention in a mammalianhost.

A wide variety of transcriptional and translational regulatory sequencesmay be employed, depending upon the nature of the host. Thetranscriptional and translational regulatory signals may be derived fromviral sources, such as adenovirus, bovine papilloma virus,cytomegalovirus, simian virus, or the like, where the regulatory signalsare associated with a particular gene sequence which has a high level ofexpression. Alternatively, promoters from mammalian expression products,such as actin, collagen, myosin, and the like, may be employed.Transcriptional initiation regulatory signals may be selected whichallow for repression or activation, so that expression of the genesequences can be modulated. Of interest are regulatory signals which aretemperature-sensitive so that by varying the temperature, expression canbe repressed or initiated, or are subject to chemical (such asmetabolite) regulation.

Expression of hOAT polypeptides of the invention in eukaryotic hostsrequires the use of eukaryotic regulatory regions. Such regions will, ingeneral, include a promoter region sufficient to direct the initiationof RNA synthesis. Preferred eukaryotic promoters include, for example,the promoter from the immediate-early gene of human cytomegalovirus(CMV) (Boshart M. et al., (1985) Cell 2:521-30, the promoter of themouse metallothionein I gene sequence (Hamer et al., (1982) J. Mol.Appl. Gen. 1:273-288); the TK promoter of Herpes virus (McKnight, (1982)Cell 31:355-365); the SV40 early promoter (Benoist et al., (1981) Nature(London) 290:304-31); and the yeast gal4 gene sequence promoter(Johnston et al., (1982) Proc. Natl. Acad. Sci. (USA) 79:6971-6975;Silver et al., (1984) Proc. Natl. Acad. Sci. (USA) 81:5951-5955).

Translation of eukaryotic mRNA is initiated at the codon which encodesthe first methionine. For this reason, it is preferable to ensure thatthe linkage between a eukaryotic promoter and a DNA sequence whichencodes an hOAT polypeptide of the invention (or a functional derivativethereof) does not contain any intervening codons which are capable ofencoding a methionine (i.e., AUG). The presence of such codons resultseither in the formation of a fusion protein (if the AUG codon is in thesame reading frame as the hOAT polypeptide of the invention codingsequence) or a frame-shift mutation (if the AUG codon is not in the samereading frame as the hOAT polypeptide of the invention coding sequence).

A nucleic acid molecule encoding an hOAT polypeptide of the inventionand an operably linked promoter may be introduced into a recipientprokaryotic or eukaryotic cell either as a nonreplicating DNA or RNAmolecule, which may either be a linear molecule or, more preferably, aclosed covalent circular molecule. Since such molecules are incapable ofautonomous replication, the expression of the gene may occur through thetransient expression of the introduced sequence. Alternatively,permanent expression may occur through the integration of the introducedDNA sequence into the host chromosome.

A vector may be employed which is capable of integrating the desiredgene sequences into the host cell chromosome. Cells which have stablyintegrated the introduced DNA into their chromosomes can be selected byalso introducing one or more markers which allow for selection of hostcells which contain the expression vector. The marker may provide forprototrophy to an auxotrophic host, biocide resistance, e.g.,antibiotics, or heavy metals, such as copper, or the like. Theselectable marker gene sequence can either be directly linked to the DNAgene sequences to be expressed, or introduced into the same cell byco-transfection. Additional elements may also be needed for optimalsynthesis of mRNA. These elements may include splice signals, as well astranscription promoters, enhancers, and termination signals. cDNAexpression vectors incorporating such elements include those describedby Okayama (1983, Mol. Cell. Biol. 3:280-289).

The introduced nucleic acid molecule can be incorporated into a plasmidor viral vector capable of autonomous replication in the recipient host.Any of a wide variety of vectors may be employed for this purpose.Factors of importance in selecting a particular plasmid or viral vectorinclude: the ease with which recipient cells that contain the vector maybe recognized and selected from those recipient cells which do notcontain the vector; the number of copies of the vector which are desiredin a particular host; and whether it is desirable to be able to“shuttle” the vector between host cells of different species.

Preferred prokaryotic vectors include plasmids such as those capable ofreplication in E. coli (such as, for example, pBR322, ColE1, pSC101,pACYC 184; “Molecular Cloning: A Laboratory Manual”, 1989, supra).Bacillus plasmids include pC194, pC221, pT127, and the like (Gryczan,In: The Molecular Biology of the Bacilli, Academic Press, NY, pp.307-329, 1982). Suitable Streptomyces plasmids include p1J101 (Kendallet al., (1987) J. Bacteriol. 169:4177-4183), Pseudomonas plasmids arereviewed in John et al. (1986) Rev. Infect. Dis. 8:693-704), and Izaki(1978) J. Bacteriol. 33:729-742).

Preferred eukaryotic plasmids include, for example, pcDNA3.1(Invitrogen), BPV, vaccinia, SV40, 2-micron circle, and the like, ortheir derivatives. Such plasmids are well known in the art (Botstein etal., (1982) Miami Wntr. Symp. 19:265-274; Broach, In: “The MolecularBiology of the Yeast Saccharomyces: Life Cycle and Inheritance”, ColdSpring Harbor Laboratory, Cold Spring Harbor, N.Y., p. 445-470, 1981;Broach, (1982) Cell 28:203-204; Bollon et al., (1980) J. Clin. Hematol.Oncol. 10:39-48; Maniatis, In: Cell Biology: A Comprehensive Treatise,Vol. 3, Gene Sequence Expression, Academic Press, NY, pp. 563-608,1980).

Once the vector or nucleic acid molecule containing the construct(s) hasbeen prepared for expression, the DNA construct(s) may be introducedinto an appropriate host cell by any of a variety of suitable means,i.e., transformation, transfection, conjugation, protoplast fusion,electroporation, particle gun technology, calciumphosphate-precipitation, direct microinjection, and the like. After theintroduction of the vector, recipient cells are grown in a selectivemedium, which selects for the growth of vector-containing cells.Expression of the cloned gene(s) results in the production of an hOATpolypeptide of the invention, or fragments thereof. This can take placein the transformed cells as such, or following the induction of thesecells to differentiate (for example, by administration ofbromodeoxyuracil to neuroblastoma cells or the like). A variety ofincubation conditions can be used to form the peptide of the presentinvention. The most preferred conditions are those which mimicphysiological conditions.

Following the introduction of the vector, cells may be allowed to growfor 1-2 days in an enriched media before they are switched to selectivemedia. The purpose of the selectable marker is to confer resistance toselection, and its presence allows growth and recovery of cells whichsuccessfully express the introduced sequences. Resistant clones ofstably transformed cells may be proliferated using tissue culturetechnique appropriate to the cell type.

Cell lines stably expressing human organic anion transporters under thecontrol of an inducible promoter can also be isolated. For inducibleexpression of human organic anion transporters, the ecdysone system fromInvitrogen (San Diego, Calif.) was used. Ecdysone is a hormone thatregulates metamorphosis in insects, such as the fruitfly Drosophila.Like other steroid hormones, ecdysone exerts its effects by binding to anuclear receptor, the VgR. The hormone-receptor complex then binds to asequence of DNA called the ecdysone response element (EcRE), andactivates transcription from promoters containing these elements. Sincemammalian cells do not express ecdysone receptors, they normally do notrespond to the insect hormone. However, if a piece of DNA under thecontrol of an ecdysone inducible promoter is transfected into amammalian cell line, treating the cell line with ecdysone will induceexpression of the said DNA.

The cloned transporters can also be expressed in the amphibian oocytes,a widely used heterologous system for the expression of membraneproteins. Complementary RNA can be synthesized from purified cDNA invitro if it is downstream of an appropriate promoter, such as the T3 orT7 phage promoter. The synthesized RNA are microinjected into each frogoocyte, which translates the message and expresses the protein in itsmembranes. Xenopus oocytes can be obtained by surgical removal ofovaries from the frog Xenopus laevis. The ovary lobes are treated withcollagenase to remove the vitalin layer. Oocytes can be maintained forup to two weeks in Barth solution (88 mM NaCl, 1 mM KCl, 2.4 mM NaHCO₃,15 mM HEPES (pH 7.6), 0.7 mM CaCl₂, 0.82 mM MgSO₄ and 50 μg/mlgentamicin) at 16° C. Typically, about 50 nl or 10-30 ng of RNA areinjected into each oocyte. The injected oocytes are maintained at 16° C.for 2-3 days and then subjected to transport assays.

Assays for Functional Expression

Assaying for functional expression of transporters can be done using alabeled compound that is a known substrate of the transporter. ForhOAT1, p-aminohippurate (PAH) is a high affinity substrate that israpidly transported. Transfected cells or injected oocytes are washedbriefly with media or Barth buffer and then incubated with the labeledPAH. After the desired incubation time, the excess label is washed awayby adding cold buffer. The cells are then solubilized with SDS and theamount of label taken up can be determined by scintillation counting.

Other methods for measuring transport activity of organic aniontransporters are well known in the art and are applicable to the presentinvention. For example, transport assays utilizing membrane vesicles wasdemonstrated by Yamazaki, et al., (1997) Drug Metabolism andDisposition, 1123-1129; and Pascolo, et al. (1998) Biochem J.331:99-103.

Cell Lines Stably Expressing hOAT Polypeptides Used to IdentifySubstrates, Inhibitors and Modulators of hOAT Polypeptides

Cell lines stably expressing hOAT polypeptides can be used to screen forother substrates or inhibitors of hOAT polypeptides through use of acompetition assay. If a test compound is well transported by an hOATprotein, it will compete with a substrate, known to be transported by aparticular hOAT, for access to the transporter. Cells expressing hOATproteins would then uptake less of the known substrate in the presencethan in the absence of the competitor.

If a test compound competes with transport of a known substrate, furthertesting is necessary to confirm that the test compound is a substrate orinhibitor. Such confirmation can be done using techniques known to thoseskilled in the art, for example, the intracellular concentration of thetest compound can be measured using HPLC. If a test compound, whichcompetes with transport of a known substrate, is also found toaccumulate inside a cell, this confirms that the test substrate is asubstrate and not an inhibitor of the particular hOAT being tested.

Modulators can be identified by adding a test compound to cell linesstably expressing hOAT polypeptides and measuring whether the additionof the test compound increases or decreases the transport of hOATsubstrates. If a modulator is identified, which decreases or inhibitsthe transport of a substrate, further testing, using techniques known tothose skilled in the art, is necessary to confirm that such modulatorsare not actually competitive inhibitors. One such technique involvesadding more substrate in a competition assay to determine if saidsubstrate can compete with the inhibitory effects of the putativemodulator. If adding more substrate has no affect on the modulatingactivity then the modulator is not a competitive inhibitor.

Nucleic Acid Probes, Methods, and Kits for Detection of the hOATPolypeptides

A nucleic acid probe of the present invention may be used to probe anappropriate chromosomal or cDNA library by usual hybridization methodsto obtain other nucleic acid molecules of the present invention. Achromosomal DNA or cDNA library may be prepared 30 from appropriatecells according to recognized methods in the art (cf. “MolecularCloning: A Laboratory Manual”, second edition, Cold Spring HarborLaboratory, Sambrook, Fritsch, & Maniatis, eds., 1989).

In the alternative, chemical synthesis can be carried out in order toobtain nucleic acid probes having nucleotide sequences which correspondto N-terminal and C-terminal portions of the amino acid sequence of thepolypeptide of interest. The synthesized nucleic acid probes may be usedas primers in a polymerase chain reaction (PCR) carried out inaccordance with recognized PCR techniques, essentially according to PCRProtocols, “A Guide to Methods and Applications”, Academic Press,Michael, et al., eds., 1990, utilizing the appropriate chromosomal orcDNA library to obtain the fragment of the present invention.

One skilled in the art can readily design such probes based on thesequence disclosed herein using methods of computer alignment andsequence analysis known in the art (“Molecular Cloning: A LaboratoryManual”, 1989, supra). The hybridization probes of the present inventioncan be labeled by standard labeling techniques such as with aradiolabel, enzyme label, fluorescent label, biotin-avidin label,chemiluminescence, and the like. After hybridization, the probes may bevisualized using known methods.

The nucleic acid probes of the present invention include RNA, as well asDNA probes, such probes being generated using techniques known in theart. The nucleic acid probe may be immobilized on a solid support.Examples of such solid supports include, but are not limited to,plastics such as polycarbonate, complex carbohydrates such as agaroseand sepharose, and acrylic resins, such as polyacrylamide and latexbeads. Techniques for coupling nucleic acid probes to such solidsupports are well known in the art.

The test samples suitable for nucleic acid probing methods of thepresent invention include, for example, cells or nucleic acid extractsof cells, or biological fluids. The samples used in the above-describedmethods will vary based on the assay format, the detection method andthe nature of the tissues, cells or extracts to be assayed. Methods forpreparing nucleic acid extracts of cells are well known in the art andcan be readily adapted in order to obtain a sample that is compatiblewith the method utilized.

One method of detecting the presence of nucleic acids of the inventionin a sample comprises (a) contacting the sample with the above-describednucleic acid probe under conditions such that hybridization occurs, and(b) detecting the presence of the probe bound to the nucleic acidmolecule. One skilled in the art would select the nucleic acid probeaccording to techniques known in the art as described above. Samples tobe tested include but should not be limited to RNA samples of humantissue.

A kit for detecting the presence of nucleic acids of the invention in asample comprises at least one container means having disposed thereinthe above-described nucleic acid probe. The kit may further compriseother containers comprising one or more of the following: wash reagentsand reagents capable of detecting the presence of bound nucleic acidprobe. Examples of detection reagents include, but are not limited toradiolabeled probes, enzymatic labeled probes (horseradish peroxidase,alkaline phosphatase), and affinity labeled probes (biotin, avidin, orstreptavidin). Preferably, the kit further comprises instructions foruse.

Antibodies, Hybridomas, Methods of Use and Kits for Detection of thehOAT Polypeptides

The present invention relates to an antibody having binding affinity toan hOAT polypeptide of the invention. The polypeptide may have the aminoacid sequence encoded by the nucleic acid sequence set forth in SEQ IDNO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6,or a functional derivative thereof, or the amino acid sequence set forthin SEQ ID NO:7 or SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11,SEQ ID NO:12, or a functional derivative thereof, or at least 9contiguous amino acids thereof (preferably, at least 20, 30, 40, 50 or60 contiguous amino acids thereof).

The present invention also relates to an antibody having specificbinding affinity to an hOAT polypeptide of the invention. Such anantibody may be isolated by comparing its binding affinity to an hOATpolypeptide of the invention with its binding affinity to otherpolypeptides. Those which bind selectively to an hOAT polypeptide of theinvention would be chosen for use in methods requiring a distinctionbetween an hOAT polypeptide of the invention and other polypeptides,including other polypeptides with similar amino acid sequences. Suchmethods could include, but should not be limited to, the analysis ofaltered hOAT polypeptide expression in tissue containing otherpolypeptides.

The hOAT polypeptides of the present invention can be used to produceantibodies or hybridomas. One skilled in the art will recognize that ifan antibody is desired, such a peptide could be generated as describedherein and used as an immunogen. The antibodies of the present inventioninclude monoclonal and polyclonal antibodies, as well fragments of theseantibodies, and humanized forms. Humanized forms of the antibodies ofthe present invention may be generated using one of the procedures knownin the art such as chimerization or CDR grafting.

The present invention also relates to hybridomas that produce theabove-described monoclonal antibodies, or binding fragment thereof. Ahybridoma is an immortalized cell line that is capable of secreting aspecific monoclonal antibody.

In general, techniques for preparing monoclonal antibodies andhybridomas are well known in the art (Campbell, “Monoclonal AntibodyTechnology: Laboratory Techniques in Biochemistry and MolecularBiology,” Elsevier Science Publishers, Amsterdam, The Netherlands, 1984;St. Groth et al., (1980), J. Immunol. Methods 35:1-21). Any animal(mouse, rabbit, and the like) which is known to produce antibodies canbe immunized with the selected polypeptide. Methods for immunization arewell known in the art. Such methods include subcutaneous orintraperitoneal injection of the polypeptide. One skilled in the artwill recognize that the amount of polypeptide used for immunization willvary based on the animal that is immunized, the antigenicity of thepolypeptide and the site of injection.

The polypeptide may be modified or administered in an adjuvant in orderto increase the peptide antigenicity. Methods of increasing theantigenicity of a polypeptide are well known in the art. Such proceduresinclude coupling the antigen with a heterologous protein (such asglobulin or β-galactosidase) or through the inclusion of an adjuvantduring immunization.

For monoclonal antibodies, spleen cells from the immunized animals areremoved, fused with myeloma cells, such as SP2/0-Ag14 myeloma cells, andallowed to become monoclonal antibody producing hybridoma cells. Any oneof a number of methods well known in the art can be used to identify thehybridoma cell that produces an antibody with the desiredcharacteristics. These include screening the hybridomas with an ELISAassay, western blot analysis, or radioimmunoassay (Lutz et al., (1988)Exp. Cell Res. 175:109-124). Hybridomas secreting the desired antibodiesare cloned and the class and subclass are determined using proceduresknown in the art (Campbell, (1984) “Monoclonal Antibody Technology:Laboratory Techniques in Biochemistry and Molecular Biology”, supra).

For polyclonal antibodies, antibody-containing antisera is isolated fromthe immunized animal and is screened for the presence of antibodies withthe desired specificity using one of the above-described procedures. Theabove-described antibodies may be detectably labeled. Antibodies can bedetectably labeled through the use of radioisotopes, affinity labels(such as biotin, avidin, and the like), enzymatic labels (such as horseradish peroxidase, alkaline phosphatase, and the like) fluorescentlabels (such as FITC or rhodamine, and the like), paramagnetic atoms,and the like. Procedures for accomplishing such labeling are well-knownin the art, for example, see Stemberger et al., (1970) J. Histochem.Cytochem. 18:315; Bayer et al., (1979) Meth. Enzym. 62:308; Engval etal., (1972) Immunol. 109:129; Goding, J. (1976) Immunol. Meth. 13:215).The labeled antibodies of the present invention can be used for invitro, in vivo, and in situ assays to identify cells or tissues thatexpress a specific peptide.

The above-described antibodies may also be immobilized on a solidsupport. Examples of such solid supports include plastics such aspolycarbonate, complex carbohydrates such as agarose and sepharose,acrylic resins such as polyacrylamide and latex beads. Techniques forcoupling antibodies to such solid supports are well known in the art(Weir et al., “Handbook of Experimental Immunology” 4th Ed., BlackwellScientific Publications, Oxford, England, Chapter 10, 1986; Jacoby etal., (1974) Meth. Enzym. 34, Academic Press, N.Y.,). The immobilizedantibodies of the present invention can be used for in vitro, in vivo,and in situ assays as well as in immunochromotography.

The present invention also encompasses a method of detecting an hOATpolypeptide in a sample, comprising: (a) contacting the sample with anabove-described antibody, under conditions such that immunocomplexesform, and (b) detecting the presence of said antibody bound to thepolypeptide. In detail, the methods comprise incubating a test samplewith one or more of the antibodies of the present invention and assayingwhether the antibody binds to the test sample.

Conditions for incubating an antibody with a test sample vary.Incubation conditions depend on the format employed in the assay, thedetection methods employed, and the type and nature of the antibody usedin the assay. One skilled in the art will recognize that any one of thecommonly available immunological assay formats (such asradioimmunoassays, enzyme-linked immunosorbent assays, diffusion basedOuchterlony, or rocket immunofluorescent assays) can readily be adaptedto employ the antibodies of the present invention. Examples of suchassays can be found in Chard (“An Introduction to Radioimmunoassay andRelated Techniques” Elsevier Science Publishers, Amsterdam, TheNetherlands, 1986), Bullock et al. (“Techniques in Immunocytochemistry,”Academic Press, Orlando, Fla. Vol. 1, 1982; Vol. 2, 1983; Vol. 3, 1985),Tijssen (“Practice and Theory of Enzyme Immunoassays: LaboratoryTechniques in Biochemistry and Molecular Biology,” Elsevier SciencePublishers, Amsterdam, The Netherlands, 1985).

The immunological assay test samples of the present invention includecells, protein or membrane extracts of cells, or biological fluids suchas blood, serum, plasma, or urine. The test samples used in theabove-described method will vary based on the assay format, nature ofthe detection method and the tissues, cells or extracts used as thesample to be assayed. Methods for preparing protein extracts or membraneextracts of cells are well known in the art and can readily be adaptedin order to obtain a sample which is testable with the system utilized.

A kit contains all the necessary reagents to carry out the previouslydescribed methods of detection. The kit may comprise: (i) a firstcontainer means containing an above-described antibody, and (ii) secondcontainer means containing a conjugate comprising a binding partner ofthe antibody and a label. Preferably, the kit also contains instructionsfor use. In another preferred embodiment, the kit further comprises oneor more other containers comprising one or more of the following: washreagents and reagents capable of detecting the presence of boundantibodies.

Examples of detection reagents include, but are not limited to, labeledsecondary antibodies, or in the alternative, if the primary antibody islabeled, the chromophoric, enzymatic, or antibody binding reagents thatare capable of reacting with the labeled antibody. The compartmentalizedkit may be as described above for nucleic acid probe kits. One skilledin the art will readily recognize that the antibodies described in thepresent invention can readily be incorporated into one of theestablished kit formats that are well known in the art.

EXAMPLES Example 1 Identification of Human EST Clones Homologous to RatOrganic Anion Transporter Proteins

The cloning of rat kidney organic anion transporter OAT1 (Sekine, etal., (1997) J. Biol. Chem. 30: 18526-18529) and liver transporter OAT2(Sekine, et al., (1998) FEBS Letters 429: 179-182) has been previouslyreported. The published amino acid sequences of OAT1 and OAT2 from ratwere used as input sequences to perform a BLAST search of humanexpressed sequence tags (EST) sequences deposited in Genbank. The BLASTsearch identified several sequences that show amino acid homology to ratOAT1 and OAT2.

EST clones accession Nos. R25797 and AI016020 show high homology to ratOAT1 and OAT2, respectively. The clones isolated using EST clones R25797and A1016020 were named hOAT1 and hOAT2, respectively. The other ESTsshow lower homology to rat OAT1 and OAT2. These other EST clones havebeen named hOAT3, hOAT4, and hOAT5. The latter three clones are notconsidered human homologues to the rat OAT1 and OAT2.

Example 2 Isolation of Full-Length Human OAT1 cDNA

The human EST clone R25797, listed in Example 1, was purchased from adistributor of IMAGE Consortium (LLNL) cDNA Clones (Lennon, et al.,Genomics 33: 151-52 (1996)). EST cDNA clone R25797 was sequenced toverify its homology to rat OAT1. The cDNA insert was excised by NotI andHindIII digestion and purified by agarose gel electrophoresis. These DNAfragments were then labeled with ³²P-α-deoxyadenosine triphosphate (3000Ci/mmol) using the Prime-a-Gene labeling system (Promega, Madison, Wis.)to a specific activity greater than 1×10⁹ dpm/μg.

A human kidney cDNA library in pTripleEx vector (Clontech, Palo Alto,Calif.) was plated out on 10 cm-diameter plates at a density of 5×10⁴pfu/plate. The plates were incubated at 37° C. for 6-8 hours until eachplaque was about 0.3-0.5 mm in diameter. Plaque lifts were performed byplacing a nylon membrane on each plate for 2 minutes. The nylon membranewas then lifted carefully from the plate and immersed in 1.5 M NaCl and0.5 M NaOH for 2 minutes to denature the attached DNA. The nylon wasthen placed in 1.5 M NaCl and 0.5 M Tris (pH 8.0) for neutralization.Finally, the membrane was quickly rinsed in 2×SSC (0.3 M NaCl and 30 mMsodium citrate). The attached DNA was crosslinked by UV irradiation.Filters were incubated in hybridization buffer with radioactive probesat a concentration of 1×10⁶ dpm/ml in roller bottles at 65° C.overnight. The next day, the filters were washed with 2×SSC and 0.1% SDSfor 15 minutes at 25° C. for two times. They were then washed with0.2×SSC and 0.1% SDS for 1 hour at 65° C. After the final wash, filterswere exposed to autoradiographic film with an intensifying screen at−80° C. overnight. Positive clones, which show up as dark spots on theautorad, were identified and a 3 mm-diameter agar plug was removed fromthe original plate.

The positive clone was purified by subsequent rounds of hybridizationuntil a well-isolated plaque could be obtained. The cDNA of interest wasexcised from the phage into a phagmid by an in vivo excision procedureusing BM25.8 cells as host (Clontech, Palo Alto, Calif.). Phagmids wereamplified, purified, and sequenced using standard procedures. The DNAsequence was analyzed for open reading frames and compared to publishedsequences.

One positive plaque isolated from the above procedure was 2.5 kb inlength. Analysis of its sequence revealed an open-reading frame with adeduced amino acid sequence showing high homology to the rat OAT1protein (Table 1). This clone was designated human OAT1 (hOAT1), or thehuman homologue of rat OAT1. The entire cDNA and deduced amino acidsequences of human OAT1 are shown in FIG. 1.

The isolated cDNA encoding for hOAT1 is predicted to encode apolypeptide with a length of 550 amino acids. Computer analysis of thededuced amino acid encoded by hOAT1 cDNA reveals consensus proteinkinase C (PKC) phosphorylation sites at amino acids 271, 278, and 284.

Computer analysis using the GAP-Alignment program from the WisconsinGenetics Software Package indicates that hOATI polypeptide is 88%identical to rat OAT1 polypeptide and 39% identical to rat OAT2polypeptide (Table 1). GAP analysis also indicates that hOATI is 86%identical to rat OAT1 nucleotide sequence (Table 1).

EST accession number R25797 is homologous to hOATI cDNA at nucleotides398-2100 (FIG. 1). TABLE 1 Homology Among Human and Rat Organic AnionTransporters hOAT1 hOAT2A hOAT2B hOAT3 hOAT4 hOAT5 N¹ (A)² N (A) N (A) N(A) N (A) N (A) rat OAT1 86% (88%) 53% (37%) 53% (37%) 60% (50%) 48%(36%) 51% (40%) rat OAT2 53% (39%) 82% (79%) 82% (79%) 51% (37%) 44%(30%) 46% (31%) hOAT1 54% (39%) 55% (39%) 61% (51%) 49% (37%) 50% (40%)hOAT2A 99% (98%) 52% (38%) 44% (32%) 45% (32%) hOAT2B 52% (39%) 44%(33%) 45% (32%) hOAT3 52% (37%) 52% (39%) hOAT4 72% (55%) hOAT5¹(N) indicates the percentage of identical nucleotides found betweencompared nucleic acid sequences as determined using the GAP-Alignmentprogram in the GCG software package, using a gap weight of 5.0 and alength weigh of 0.3.²(A) indicates the percentage of identical amino acids found betweencompared polypeptides as determined using the GAP-Alignment program inthe GCG software package, using a gap weight of 3.0 and a length weightof 0.1.

Example 3 Isolation of Full-Length Human OAT2A and OAT2B cDNA

Human OAT2 cDNA was cloned in the same manner as described for humanOAT1 cDNA in Example 1 except that EST accession number-A1016020 wasused as a probe. In addition, hOAT2A and hOAT2B were isolated from acDNA library obtained from human liver rather than kidney, as was hOAT1.

The entire cDNA and deduced amino acid sequences of human OAT2A areshown in FIG. 2. Human OAT2A (hOAT2A) cDNA is predicted to encode apolypeptide that is 546 amino acids in length. Computer analysis of thededuced amino acid sequence using the GAP-Alignment program indicatesthat human OAT2A polypeptide is 79% identical to rat OAT2 polypeptide(Table 1). GAP analysis also indicates that the nucleotide sequence forhOAT2A is 82% identical to the nucleotide sequence of rat OAT2.

EST accession number A 1016020 is homologous to hOAT2A cDNA atnucleotide sequences 20-1400 (FIG. 2).

The entire cDNA and deduced amino acid sequences of human OAT2B (hOAT2B)are shown in FIG. 3. Human OAT2B cDNA is predicted to encode apolypeptide that is 538 amino acids in length. Human OAT2B is identicalto hOAT2A except at its C-terminus end. Without wishing to be bound byany particular theory, hOAT2B could be an mRNA sp1ice variant of hOAT2A.

Example 4 Isolation of Full-Length Human OAT3 cDNA

Human OAT3 (hOAT3) cDNA was isolated as described for hOAT1 cDNA inExample 1 except that EST accession number-H41333 was used as a probe.The entire cDNA and deduced amino acid sequences of human OAT3 are shownin FIG. 4. Computer analysis of the hOAT3 cDNA sequence predicts thathOAT3 cDNA encodes a polypeptide that is 542 amino acids in length.Computer analysis of the deduced amino acid sequence reveals consensusPKC phosphorylation sites at amino acids 259, 266, 269, 511, and 527.

EST accession number H41333 is homologous to hOAT3 cDNA at nucleotides1169-2121 (FIG. 4).

Example 5 Isolation of Full-Length Human OAT4 cDNA

Human OAT4 (hOAT4) cDNA was cloned in the same manner as described forhuman OAT1 cDNA in Example 1 except that EST accession number-AA705512was used as a probe. In addition, hOAT4 cDNA was isolated from a cDNAlibrary obtained from human liver rather than kidney, as was hOAT1. Theentire cDNA and deduced amino acid sequences of human OAT4 are shown inFIG. 5. Computer analysis of human OAT4 cDNA predicts that hOAT4 cDNAencodes a polypeptide that is 554 amino acids in length. Computeranalysis of the deduced amino acid sequence reveals a consensus PKCphosphorylation site at amino acid 324.

EST accession number AA705512 is homologous to hOAT4 cDNA at nucleotides1-1232 (FIG. 5).

Example 6 Isolation of Full-Length hOAT5 cDNA

Human OAT5 (hOAT5) cDNA was cloned in the same manner as described forhuman OAT1 cDNA in Example 1 except that EST accession number-AAA705512was used as a probe. In addition, hOAT5 cDNA was isolated from a cDNAlibrary obtained from human liver rather than kidney, as was hOAT1. Theentire cDNA and deduced amino acid sequences of human OAT5 are shown inFIG. 6. Human OAT5 cDNA is predicted to encode a polypeptide that is 541amino acids in length. Computer analysis of the deduced amino acidsequence reveals consensus PKC phosphorylation sites at amino acids 282,289, 345, and 526.

EST accession number AA705512 is homologous to hOAT5 cDNA at nucleotides1-1193 (FIG. 6).

Example 7 Functional Expression of Human OAT1 in HeLa Cells

Human OAT1 cDNA was subcloned into the mammalian expression vectorpcDNA3.1 (Invitrogen, San Diego, Calif.), which contains acytomegalovirus promoter. The plasmid construct (pcDNA-hOAT1) was thenamplified and purified. The construct was transfected into HeLa cells(ATCC), cultured in 25 mm² flasks, using the Effectene transfectionreagent (Qiagen, Stanford Santa Clarita, Calif.). Control cell lineswere transfected with a pcDNA-lacZ construct. One day post-transfection,the cells were split into six (6)-well plates and cultured for anothertwenty-four (24) hours. Transfected cells were tested for transport of³H-p-aminohippurate, a classical substrate of the kidney organic aniontransporter. Since OAT1 is an antiporter, the transfected cells werefirst preloaded with 1 mM glutarate for one (1) hour. Theglutarate-containing media was removed and replaced with mediacontaining 50 μM PAH at 1 μCi/ml. After ten (10) minutes at 37° C., themedia was aspirated and the cells were washed three times with ice coldphosphate-buffered saline (PBS). The cells were then solubilized with0.5% SDS. The amount of labeled PAH transported into the cells wasdetermined by a scintillation counter. As shown in FIG. 7, HeLa cellstransfected with hOAT1 were able to uptake PAH whereas HeLa cellstransfected with a vector control were only able to uptake a very smallamount of PAH (almost 10-fold less than cells transfected with a vectorcontaining DNA encoding hOAT1).

Example 8 Functional Expression of hOAT 1 in Xenopus Oocytes

Complementary hOATI RNA was synthesized in vitro from the pcDNA-hOAT1plasmid using T7 RNA polymerase (Ambion, Austin, Tex.). Thetranscription reaction was performed at 37° C. for 2 hours. The RNA waspurified by phenol and chloroform extraction and precipitated withammonium acetate and ethanol. Purified RNA was analyzed for size andpurity by agarose gel electrophoresis. RNA was resuspended in water at aconcentration of 0.3 μg/μl. Oocytes were prepared from ovaries removedfrom female Xenopus laevis according to White et al., (1985) ^(PNAS)82:4852-56. About 15 ng of hOAT1 cRNA in 50 nl was injected into eachoocyte. As a control, 50 nl of water was injected into each oocyte. Theinjected oocytes were assayed for transport of PAH two (2) dayspost-injection. Oocytes were preloaded with 1 mM glutarate for 2 hoursin Barth buffer. Transport assays were conducted in modified Barthbuffer (100 mM NaCl, 2 mM KCl, 1 mM CaCl₂, 1 mM MgCl₂, 10 mM HEPES, pH7.5) with 50 μM PAH at 4 μCi/ml. After a one (1) hour incubation at 25°C., oocytes were washed with 3 ml ice cold transport assay buffer three(3) times. Each oocyte was solubilized in 0.5 ml 0.5% SDS and subjectedto scintillation counting. As shown in FIG. 8, oocytes injected with thehOAT1 RNA uptake ³H-PAH to a much greater extent than water injectedoocytes.

Example 9 Identification of Fluorescein as a Substrate for hOAT1

Human embryonic kidney (HEK293) cells were transfected with pcDNA-hOAT1,as in Example 7. One day after transfection the cells were assayed fortransport of fluorescein or SITS. Ten (10) μM of either substrate wasadded to the external media for ten (10) minutes at 37° C. The cellswere washed with ice-cold PBS to remove excess label and analyzed undera fluorescent microscope. Cells that take up either fluorescein or SITSare predicted to turn fluorescent green. Fluorescent microscopy revealedthat hOAT1-expressing cells took up fluorescein but not SITS.

Example 10 Competition Assay to Identify Substrates of hOAT1 UsingFluorescein

HEK293 cells were transiently transfected with pcDNA3-hOAT1 as inExample 7 and assayed for transport of fluorescein, but in the presenceor absence of 0.5 mM PAH. Since fluorescein and PAH are both substratesof hOAT1, they will compete for access to the transporter. As evidencedby fluorescent microscopy, hOAT1-expressing cells showed uptake offluorescein in the absence but not in the presence of external PAH.

Example 11 Establishment of Cell Lines Stably Expressing hOATs

Vector pIND(sp1) (Invitrogen) contains the ecdysone-inducible promoter(EcRE). Using standard methods known to those in the art, cDNAs encodinghOAT1, hOAT2A, into vector pIND/sp1 downstream of its EcRE promoter.After cloning, the vectors were separately transfected into cell lineEcR293 (Invitrogen). The EcR293 cell line is a derivative of cell lineHEK 293 and contains a stably integrated pVgRXR vector. The pVgRXRvector constitutively expresses the ecdysone receptor and the retinoid Xreceptor (RXR). The ecdysone receptor and RXR form a heterodimer which,upon activation by the hormone ecdysone, binds to a promoter containingthe EcRE. The pVgRXR vector was maintained within the EcR293 cell lineby selection with 400 μg/ml zeocin. The pIND(sp1)-hOAT plasmids containa neomycin resistance marker, and cells stably integrating the plasmidswere selected for by culturing the cells in media supplemented with 400μg/ml G418. After two weeks of selection, individual colonies appearedand were isolated and expanded. To test for expression of thetransporter, the cells were induced with 1.25 μg/ml ponasterone, anecdysone analogue, for twenty-four (24) hours. The cells were thensubjected to standard transport assays.

EcR293 cells with stably integrated pIND(sp1)-hOAT1 expressed hOAT1 at abasal level that was not harmful to the cells. These cells grow at arapid rate comparable to the parental EcR293 cells. Hence, the hOAT1cDNA can be maintained within these cells indefinitely. Expression ofhOAT1 at high levels can be easily achieved by inducing with ponasteronefor twenty-four (24) hours. As shown in FIG. 9, ECR293 cells containinghOAT1 in plasmid pIND(sp-1) uptake approximately 50-fold more PAH wheninduced by ponasterone than non-induced cells containingpIND(sp-1)-hOAT1. ECR293 cells stably containing hOAT2A, in plasmidpIND(sp-1), uptake approximately 2.6-fold more ³H-PAH when induced byponasterone than non-induced cells (FIG. 10). Cell lines stablyexpressing hOAT2B, hOAT3, hOAT4, and hOAT5 can be established using thesame method as for hOAT1 and hOAT2A.

Example 12 Competition Assay to Identify New Substrates of hOAT2, Usinga Radiolabeled Substrate

EcR293/OAT2A cells were assayed for transport of ³H-PAH as in Example 7.The uptake of ³H-PAH was blocked by 100 μM sulfobromophthalein (BSP) butnot 500 μM α-ketoglutarate (AKG) (FIG. 11). The competition assay thusidentified BSP and not AKG as a preferred substrate of hOAT2A.

Example 13 Predicting Pharmacokinetics in Man Using a Panel of hOATExpressing Cells

A panel of different cell lines, each expressing a particular human OATprotein, can be used to predict pharmacokinetics for anionic drugs. Forinstance, PAH is a compound transported rapidly by hOAT1 and to a muchlesser extent by the other human OATs disclosed in the invention. Thepanel of hOAT expressing cells would predict that PAH is eliminatedrapidly by the human kidney, as previously described. Similarly, thecompetition assays in Examples 10 and 12 showed that fluorescein is avery good substrate of human OAT1 and that BSP is a good substrate ofhuman OAT2A. Therefore, the assay would predict that fluorescein iseliminated from the human body mainly via kidney excretion and that BSPis mainly eliminated from the human body via liver excretion.

Further confirmation that a compound is a substrate of an hOAT would bedone using in vivo tissue distribution assays. Compounds found to betransported by hOATs, using the cell line described above, would beinjected into animals and followed to confirm their transport by theappropriate hOAT. For instance, a compound found to be transported by acell line stably expressing hOAT1, would be expected to be excreted inthe urine of an animal injected with the said compound. Similarly, acompound found to be transported by a cell line expressing hOAT2, wouldbe expected to be transported into the liver of animals injected withsaid compound.

Since hOAT4 and 5 have homology to OCTs, their range of of substratesmay include organic cations. Therefore, these transporters may be usedto predict the pharmacokinetics of certain cationic drugs as well, usingthe methods described herein.

Example 14 Identification of hOAT Substrates Using Antiporter Activity

EcR293/hOAT1 cells are incubated with ³H-α-ketoglutarate for an hour topreload with labeled α-ketoglutarate. A test substrate is added to thecells. After a 10 minute incubation, an aliquot of the supernatant isremoved and measured for effluxed α-ketoglutarate by scintillationcounting. Substrates of hOAT1 will show trans-stimulation ofα-ketoglutarate exchange. Inhibitors or non-substrates of hOAT1 will notstimulate α-ketoglutarate efflux.

Example 15 Cytoxicity Assays Using Cell Lines Stably Expressing hOATPolypeptides

EcR293 cells stably transformed with hOAT polypeptides can be used toscreen for compounds potentially toxic to certain organs or tissuesthrough use of a cytoxicity assay. Test compounds can be added to celllines stably expressing hOAT polypeptides. Compounds transported by hOATpolypeptides and which are toxic will cause cell death. Cell death canbe measured or determined using techniques well known in the art, suchas visual inspection, microscopic inspection, and dye exclusion. Usingdye exclusion, healthy cells are able to exclude a particular dyewhereas injured or dead cells are unable to exclude the dye.

The foregoing examples are not limiting and are merely representative ofvarious aspects and features of the present invention. All referencesreferred to above are incorporated herein by reference.

Example 16 Functional Expression of hOAT3 in Xenopus Oocytes

Complementary hOAT3 RNA was synthesized in vitro from the pcDNA-hOAT3plasmid using T7 RNA polymerase (Ambion, Austin, Tex.). Thetranscription reaction was performed at 37° C. for 2 hours. The RNA waspurified by phenol and chloroform extraction and precipitated withammonium acetate and ethanol. Purified RNA was analyzed for size andpurity by agarose gel electrophoresis. RNA was resuspended in water at aconcentration of 0.5 μg/μl. Oocytes were prepared from ovaries removedfrom female Xenopus laevis according to White et al., (1985) PNAS82:4852-56. About 25 ng of hOAT3 cRNA in 50 nl was injected into eachoocyte. As a control, 50 nl of water was injected into each oocyte. Theinjected oocytes were assayed for transport of PAH two (2) dayspost-injection. Transport assays were conducted in modified Barth buffer(100 mM NaCl, 2 mM KCl, 1 mM CaCl₂, 1 mM MgCl₂, 10 mM HEPES, pH 7.5)with 50 μM PAH at 10 μCi/ml. After a one (1) hour incubation at 25° C.,oocytes were washed with 3 ml ice cold transport assay buffer three (3)times. Each oocyte was solubilized in 0.5 ml 0.5% SDS and subjected toscintillation counting. As shown in FIG. 12, oocytes injected with thehOAT3 RNA uptake ³H-PAH to a much greater extent than water injectedoocytes.

Example 17 Determination of Tissue Distribution of hOATs

The individual hOAT cDNAs were labeled with ³2P-ATP to a specificactivity of >1×10⁹ dpm/μg using the Strip-EZ kit™ (Ambion). The Strip-EZkit™ allows easy removal of probes from a blot and the blot can thus bereprobed several times without substantial loss of signal. The HumanMultiple Tissue Northern blot was obtained from Clontech, whichcontained mRNA extracted from 12 different human tissues. The blot wassuccessively hybridized with each hOAT probe in 5 ml of ExpressedHybbuffer (Clontech) at a final concentration of 3-5×10⁶ dpm/ml.Hybridization was carried out at 65° C. for 4 hrs. The blot was washedtwo times with 2×SSC and 0.1%/SDS at 65° C. for 15 min each, and thenonce with 0.1×SSC and 0.1% SDS at 65° C. for 1 hr. The blot was thenexposed to BioMax MR autographic film (Kodak) with intensifying screenat −80° C. for 24 hrs.

The northern analysis shows that hOAT1 was expressed only in humankidney (FIG. 13). The OAT1 probe hybridized to a major band at about 2.4kb. The hOAT2 probe hybridized to two major bands at about 2.4 and about3 kb from liver and kidney, with higher levels in the liver than kidney(FIG. 13). HOAT3 was expressed only in the kidney, with a majortranscript of about 2.4 kb and minor transcript at about 4.3 kb (FIG.13). HOAT4 and hOAT5 were expressed exclusively in the liver (FIG. 13).hOAT4 probe hybridized to a major band at about 4.3 kb and hOAT5 probehybridized to a major band at about 2.5 kb. Hence, northern analysesindicated that hOAT1 and 3 are mainly kidney transporters and hOAT4 and5 are liver transporters, whereas hOAT2 is a liver and kidneytransporter.

All patents and publications mentioned in the specification areindicative of the levels of skill of those skilled in the art to whichthe invention pertains. All references cited in this disclosure areincorporated by reference to the same extent as if each reference hadbeen incorporated by reference in its entirety individually. None of thereferences are admitted to be prior art.

One skilled in the art would readily appreciate that the presentinvention is well adapted to carry out the objects and obtain the endsand advantages mentioned, as well as those inherent therein. The methodsand compositions described herein as presently representative ofpreferred embodiments are exemplary and are not intended as limitationson the scope of the invention. Changes therein and other uses will occurto those skilled in the art, which are encompassed within the spirit ofthe invention, are defined by the scope of the claims.

It will be readily apparent to one skilled in the art that varyingsubstitutions and modifications may be made to the invention disclosedherein without departing from the scope and spirit of the invention.Thus, such additional embodiments are within the scope of the presentinvention and the following claims.

The invention illustratively described herein suitably may be practicedin the absence of any element or elements, limitation or limitationswhich is not specifically disclosed herein. Thus, for example, in eachinstance herein any of the terms “comprising”, “consisting essentiallyof” and “consisting of” may be replaced with either of the other twoterms. The terms and expressions which have been employed are used asterms of description and not of limitation, and there is no intentionthat in the use of such terms and expressions of excluding anyequivalents of the features shown and described or portions thereof, butit is recognized that various modifications are possible within thescope of the invention claimed. Thus, it should be understood thatalthough the present invention has been specifically disclosed bypreferred embodiments, optional features, modification and variation ofthe concepts herein disclosed may be resorted to by those skilled in theart, and that such modifications and variations are considered to bewithin the scope of this invention as defined by the description and theappended claims.

In addition, where features or aspects of the invention are described interms of Markush groups or other grouping of alternatives, those skilledin the art will recognize that the invention is also thereby describedin terms of any individual member or subgroup of members of the Markushgroup or other group.

Other embodiments are within the following claims.

1-33. (canceled)
 34. An isolated, enriched, or purified human OATpolypeptide encoded by the nucleic acid sequence set forth in SEQ IDNO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, or SEQ ID NO:6, or theamino acid sequence set forth in SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10,SEQ ID NO:11, or SEQ ID NO:12.
 35. The polypeptide of claim 34, whereinsaid polypeptide is a fragment of the polypeptide encoded by the nucleicacid sequence set forth in SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ IDNO:5, or SEQ ID NO:6, or the amino acid sequence set forth in SEQ IDNO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, or SEQ ID NO:12.
 36. Thepolypeptide of claim 34, wherein said polypeptide is isolated, purified,or enriched from a cell that comprises an endogenous nucleic acidmolecule that encodes said polypeptide.
 37. The polypeptide of claim 34,wherein said polypeptide is isolated, purified, or enriched from a cellthat is transformed with a nucleic acid molecule that encodes saidpolypeptide.
 38. The polypeptide of claim 34, wherein said polypeptideis chemically synthesized. 39-54. (canceled)